Stabilized Angiopoietin-2 Antibodies And Uses Thereof

ABSTRACT

Stabilized antibodies directed to Angiopoeitin-2 and uses of such antibodies are described. Nucleic acid and amino acid sequences, hybridomas or other cell lines for expressing such antibodies are also provided.

I. FIELD

The invention relates to stabilized monoclonal antibodies againstAngiopoietin-2 (Ang-2) and uses of such antibodies. Aspects of theinvention also relate to hybridomas or other cell lines expressing suchantibodies. The described antibodies are useful as diagnostics and forthe treatment of diseases associated with the activity of Ang-2 and/orAng-1.

II. BACKGROUND

Angiogenesis is the process of forming new capillaries from preexistingblood vessels and is an essential component of embryogenesis, normalphysiological growth, repair, and tumor expansion. Although a variety offactors can modulate endothelial cell (EC) responses in vitro and bloodvessel growth in vivo, only vascular endothelial growth factor (VEGF)family members and the angiopoietins are believed to act almostexclusively on vascular ECs (Yancopoulos et al., Nature 407:242-48(2000)).

The angiopoietins were discovered as ligands for the Ties, a family oftyrosine kinases that is selectively expressed within the vascularendothelium (Yancopoulos et al., Nature 407:242-48 (2000)). There arenow four definitive members of the angiopoietin family: Angiopoietin-3and -4 (Ang-3 and Ang-4) may represent widely diverged counterparts ofthe same gene locus in mouse and man (Kim et al., FEBS Let, 443:353-56(1999); Kim et al., J Biol Chem 274:26523-28 (1999)). Ang-1 and Ang-2were originally identified in tissue culture experiments as agonist andantagonist, respectively (Davis et al., Cell 87:1161-69 (1996);Maisonpierre et al., Science 277:55-60 (1997)). All of the knownangiopoietins bind primarily to Tie2, and both Ang-1 and -2 bind to Tie2with an affinity of 3 nM (Kd) (Maisonpierre et al., Science 277:55-60(1997)). Ang-1 was shown to support EC survival and to promoteendothelium integrity, (Davis et al., Cell 87:1161-69 (1996); Kwak etal., FEBS Lett 448:249-53 (1999); Suri et al., Science 282:468-71(1998); Thurston et al., Science 286: 2511-14 (1999); Thurston et al.,Nat. Med. 6:460-63 (2000)), whereas Ang-2 had the opposite effect andpromoted blood vessel destabilization and regression in the absence ofthe survival factors VEGF or basic fibroblast growth factor(Maisonpierre et al., Science 277:55-60 (1997)). However, many studiesof Ang-2 function have suggested a more complex situation. Ang-2 mightbe a complex regulator of vascular remodeling that plays a role in bothvessel sprouting and vessel regression. Supporting such roles for Ang-2,expression analyses reveal that Ang-2 is rapidly induced, together withVEGF, in adult settings of angiogenic sprouting, whereas Ang-2 isinduced in the absence of VEGF in settings of vascular regression(Holash et al., Science 284:1994-98 (1999); Holash et al., Oncogene18:5356-62 (1999)). Consistent with a context-dependent role, Ang-2binds to the same endothelial-specific receptor, Tie-2, which isactivated by Ang-1, but has context-dependent effects on its activation(Maisonpierre et al., Science 277:55-60 (1997)).

Corneal angiogenesis assays have shown that both Ang-1 and Ang-2 hadsimilar effects, acting synergistically with VEGF to promote growth ofnew blood vessels (Asahara et al., Circ. Res. 83:233-40 (1998)). Thepossibility that there was a dose-dependent endothelial response wasraised by the observation that in vitro at high concentration, Ang-2 canalso be pro-angiogenic (Kim et al., Oncogene 19:4549-52 (2000)). At highconcentration, Ang-2 acts as an apoptosis survival factor forendothelial cells during serum deprivation apoptosis through activationof Tie2 via PI-3 kinase and Akt pathway (Kim et al., Oncogene 19:4549-52(2000)).

Other in vitro experiments suggested that during sustained exposure, theeffects of Ang-2 may progressively shift from that of an antagonist toan agonist of Tie2, and at later time points, it may contribute directlyto vascular tube formation and neovessel stabilization(Teichert-Kuliszewska et al., Cardiovasc. Res. 49:659-70 (2001)).Furthermore, if ECs were cultivated on fibrin gel, activation of Tie2with Ang-2 was also observed, perhaps suggesting that the action ofAng-2 could depend on EC differentiation state (Teichert-Kuliszewska etal., Cardiovasc. Res. 49:659-70 (2001)). In microvascular EC cultured ina three-dimensional collagen gel, Ang-2 can also induce Tie2 activationand promote formation of capillary-like structures (Mochizuki et al., J.Cell. Sci. 115:175-83 (2002)). Use of a 3-D spheroidal coculture as anin vitro model of vessel maturation demonstrated that direct contactbetween ECs and mesenchymal cells abrogates responsiveness to VEGF,whereas the presence of VEGF and Ang-2 induced sprouting (Korff et al.,Faseb J. 15:447-57 (2001)). Etoh et al. demonstrated that ECs thatconstitutively express Tie2, the expression of MMP-1, -9 and u-PA werestrongly up-regulated by Ang-2 in the presence of VEGF (Etoh, et al.,Cancer Res. 61:2145-53 (2001)). With an in vivo pupillary membranemodel, Lobov et al. showed that Ang-2 in the presence of endogenous VEGFpromotes a rapid increase in capillary diameter, remodeling of the basallamina, proliferation and migration of endothelial cells, and stimulatessprouting of new blood vessels (Lobov et al., Proc. Natl. Acad. Sci. USA99:11205-10 (2002)). By contrast, Ang-2 promotes endothelial cell deathand vessel regression without endogenous VEGF (Lobov et al., Proc. Natl.Acad. Sci. USA 99:11205-10 (2002)). Similarly, with an in vivo tumormodel, Vajkoczy et al. demonstrated that multicellular aggregatesinitiate vascular growth by angiogenic sprouting via the simultaneousexpression of VEGFR-2 and Ang-2 by host and tumor endothelium (Vajkoczyet al., J. Clin. Invest. 109:777-85 (2002)). This model illustrated thatthe established microvasculature of growing tumors is characterized by acontinuous remodeling, putatively mediated by the expression of VEGF andAng-2.

Knock-out mouse studies of Tie-2 and Angiopoietin-1 show similarphenotypes and suggest that Angiopoietin-1 stimulated Tie-2phosphorylation mediates remodeling and stabilization of developingvessel, promoting blood vessel maturation during angiogenesis andmaintenance of endothelial cell-support cell adhesion (Dumont et al.,Genes & Development, 8:1897-1909 (1994); Sato, Nature, 376:70-74 (1995);(Thurston, G. et al., 2000 Nature Medicine: 6, 460-463)). The role ofAngiopoietin-1 is thought to be conserved in the adult, where it isexpressed widely and constitutively (Hanahan, Science, 277:48-50 (1997);Zagzag, et al., Exp Neurology, 159:391-400 (1999)). In contrast,Angiopoietin-2 expression is primarily limited to sites of vascularremodeling where it is thought to block the constitutive stabilizing ormaturing function of Angiopoietin-1, allowing vessels to revert to, andremain in, a plastic state which may be more responsive to sproutingsignals (Hanahan, 1997; Holash et al., Oncogene 18:5356-62 (1999);Maisonpierre, 1997). Studies of Angiopoietin-2 expression inpathological angiogenesis have found many tumor types to show vascularAngiopoietin-2 expression (Maisonpierre et al., Science 277:55-60(1997)). Functional studies suggest Angiopoietin-2 is involved in tumorangiogenesis and associate Angiopoietin-2 overexpression with increasedtumor growth in a mouse xenograft model (Ahmad, et al., Cancer Res.,61:1255-1259 (2001)). Other studies have associated Angiopoietin-2overexpression with tumor hypervascularity (Etoh, et al., Cancer Res.61:2145-53 (2001); Tanaka et al., Cancer Res. 62:7124-29 (2002)).

In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2 have beenproposed as possible anti-cancer therapeutic targets (See, for example,U.S. Pat. Nos. 6,166,185, 5,650,490, 5,814,464, US Patent PublicationNo. 20060018909 and PCT publication Nos. WO2006/068953 andWO2007/068895).

Ang-2 is expressed during development at sites where blood vesselremodeling is occurring (Maisonpierre et al., Science 277:55-60 (1997)).In adult individuals, Ang-2 expression is restricted to sites ofvascular remodeling as well as in highly vascularized tumors, includingglioma (Osada et al., Int. J. Oncol. 18:305-09 (2001); Koga et al.,Cancer Res. 61:6248-54 (2001)), hepatocellular carcinoma, (Tanaka et al,J. Clin. Invest. 103:341-45 (1999)), gastric carcinoma, (Etoh, et al.,Cancer Res. 61:2145-53 (2001); Lee et al, Int. J. Oncol. 18:355-61(2001)), thyroid tumor (Bunone et al., Am J Pathol 155:1967-76 (1999)),non-small cell lung cancer (Wong et al., Lung Cancer 29:11-22 (2000)),cancer of colon (Ahmad et al., Cancer 92:1138-43 (2001)), and prostateWurmbach et al., Anticancer Res. 20:5217-20 (2000)). Some tumor cellsare found to express Ang-2. For example, Tanaka et al. (1999) detectedAng-2 mRNA in 10 out of 12 specimens of human hepatocellular carcinoma(HCC). Ellis' group reported that Ang-2 is expressed ubiquitously intumor epithelium (Ahmad et al., Cancer 92:1138-43 (2001)). Otherinvestigators reported similar findings (Chen et al., J. Tongji Med.Univ. 21:228-30, 235 (2001)). By detecting Ang-2 mRNA levels in archivedhuman breast cancer specimens, Sfilogoi et al. (Int. J. Cancer103:466-74 (2003)) reported that Ang-2 mRNA is significantly associatedwith auxiliary lymph node invasion, short disease-free time and pooroverall survival. Tanaka et al. (Cancer Res. 62:7124-29 (2002) revieweda total of 236 patients of non-small cell lung cancer (NSCLC) withpathological stage-I to -IIIA, respectively. Using immunohistochemistry,they found that 16.9% of the NSCLC patients were Ang-2 positive. Themicrovessel density for Ang-2 positive tumor is significantly higherthan that of Ang-2 negative. Such an angiogenic effect of Ang-2 was seenonly when VEGF expression was high. Moreover, positive expression ofAng-2 was a significant factor to predict a poor postoperative survival.However, they found no significant correlation between Ang-1 expressionand the microvessel density (Tanaka et al., Cancer Res. 62:7124-29(2002)). These results suggest that Ang-2 is an indicator of poorprognosis patients with several types of cancer.

The development of antibody therapeutics for the treatment of disease isa complex process in which candidate molecules must pass throughmultiple tests to ensure suitability in every application. In mostcases, the initial candidates are developed based on a pre-determinedgroup of desired characteristics, such as antigen affinity, antibodyformat, and others. Once a candidate molecule is chosen, the suitabilityfor large scale production and stability are considered. Often, thecandidate molecule, although highly applicable based on initial desiredcharacteristics, needs to be refined to ensure the prolonged stabilityand high production efficiency required for feasibility as a commercialtherapeutic.

Disulfide bond formation in proteins is a complex process, which isdetermined by the redox potential of the environment and specializedthiol-disulfide exchanging enzymes (Creighton, Methods Enzymol. 107,305-329, 1984; Houee-Levin, Methods Enzymol. 353, 35-44, 2002). Thedisulfides are formed in cells during or shortly after secretion of thenascent chains into the endoplasmic reticulum. Several conformationalisoforms of the same protein, but with different disulfide structures,can be generated during recombinant protein production in mammaliancells due to the failing disulfide formation process, close proximity ofcysteine residues in the protein structure or surface exposure ofunpaired cysteine residues.

In general, cysteine residues in proteins (for example, antibodiesspecific for Ang-2) are either engaged in cysteine-cysteine disulfidebonds or sterically protected from the disulfide bond formation whenthey are a part of folded protein region. When a cysteine residue doesnot have a pair in protein structure and is not sterically protected byfolding, it can form a disulfide bond with a free cysteine from solutionin a process known as disulfide shuffling. In another process known asdisulfide scrambling, free cysteines may also interfere with naturallyoccurring disulfide bonds (such as those present in antibody structures)and lead to low binding, low biological activity and/or low stability.

Glycosylation of immunoglobulins has also been shown to have significanteffects on their binding characteristics, effector functions, structuralstability, and rate of secretion from antibody-producing cells(Leatherbarrow et al., Mol. Immunol. 22:407 (1985)). In particular,glycosylation of the variable region of antibodies may influence theinteraction of the antibody with its cognate antigen. It has been shownthat glycosylation in the variable region can have a negative effect onantibody binding affinity, likely due to steric hindrance (Co, M. S., etal., Mol. Immunol. (1993) 30:1361-1367). The heterogeneity of theglycosylation process may also lead to a number of antibody species withaltered binding properties. As such, it is desirable to remove or alterthe interfering glycosylation site to ensure a consistent antigenbinding profile.

Thus, there is a need to develop highly stable antibodies specific forAng-2 for a variety of therapeutic and diagnostic applications.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

III. SUMMARY

One aspect of the invention provides certain antibodies directed toAngiopoietin-2 (hereinafter referred to as “antibodies of theinvention”) which are stable and do not readily aggregate in certainpharmaceutical formulations.

In one embodiment, an Ang-2 antibody is provided which comprises a lightchain shown as MEDI1, MEDI2, MEDI3, MEDI6 or MEDI4 and/or a heavy chainshown as MEDI5.

Antibodies of the invention have the ability to specifically bind Ang-2and inhibit tumor angiogenesis and reduce tumor growth. Mechanisms bywhich this can be achieved can include, but are not limited to, eitherinhibition of binding of Ang-2 and/or Ang1 to its receptor Tie2,inhibition of Ang-2 and/or Ang-1 induced Tie2 signaling, inhibition ofAng-2 and/or Ang-1 induced Tie2 phosphorylation, or increased clearanceof Ang-2 and/or Ang1, therein reducing the effective concentration ofAng-2 and/or Ang-1.

In one embodiment, the antibodies of the invention exhibit enhancedstability as compared to the control Ang-2 specific antibody 3.19.3. Inanother embodiment, the antibodies of the invention exhibit enhancedproduction yields as compared to a control Ang-2 specific antibody. Inone embodiment, the antibody of the invention is a human Ang-2 antibodywhich comprises a Val substitution at position 37 of the heavy chain asdefined by the EU numbering system ((Kabat et al., Sequences of Proteinsof Immunological Interest, Fifth Edition, NIH Publication 91-3242,Bethesda Md. (1991), vols. 1-3.), and a substitution of Asp, Thr, Asn,or Ala at position 49 of the light chain based on the Kabat numberingsystem.

In another embodiment, the antibodies of the invention may comprise avariable light chain amino acid sequence selected from the groupconsisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ IDNo.:5), MEDI6 (SEQ ID No.:8) and MEDI4 (SEQ ID No.:6). In anotherembodiment, the antibodies of the invention may comprise the heavy chainvariable amino acid sequence MEDI5 (SEQ ID No.:7). In anotherembodiment, the antibodies of the invention may comprise a variablelight chain amino acid sequence selected from the group consisting ofMEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID No.:5), MEDI6(SEQ ID No.:8) and MEDI4 (SEQ ID No.:6) as well as a heavy chainvariable sequence defined as MEDI5 (SEQ ID No.:7).

In another aspect, the invention also provides nucleic acid sequences,vectors and cell lines for expression of the antibodies of theinvention.

The invention further provides methods for assaying the level ofAngiopoietin-2 (Ang-2) in a patient sample, comprising contacting ananti-Ang-2 antibody with a biological sample from a patient, anddetecting the level of binding between said antibody and Ang-2 in saidsample. In more specific embodiments, the biological sample is blood.

In other embodiments the invention provides compositions, including anantibody or functional fragment thereof, and a pharmaceuticallyacceptable carrier.

Further embodiments include methods of effectively treating an animalsuffering from an angiogenesis-related disease, including selecting ananimal in need of treatment for a neoplastic or non-neoplastic disease,and administering to said animal a therapeutically effective dose of amonoclonal antibody of the invention.

Treatable angiogenesis-related diseases can include neoplastic diseases,such as, melanoma, small cell lung cancer, non-small cell lung cancer,glioma, hepatocellular (liver) carcinoma, thyroid tumor, gastric(stomach) cancer, prostate cancer, breast cancer, ovarian cancer,bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidneycancer, colon cancer, pancreatic cancer, esophageal carcinoma, head andneck cancers, mesothelioma, sarcomas, biliary (cholangiocarcinoma),small bowel adenocarcinoma, pediatric malignancies and epidermoidcarcinoma.

Additional embodiments include methods of inhibiting Angiopoietin-2(Ang-2) induced angiogenesis in an animal. These methods includeselecting an animal in need of treatment for Ang-2 induced angiogenesis,and administering to said animal a therapeutically effective dose of anantibody of the invention.

Further embodiments include the use of an antibody of the invention inthe preparation of medicament for the treatment of angiogenesis-relateddiseases in an animal, wherein said antibody specifically binds toAngiopoietin-2 (Ang-2). Treatable angiogenesis-related diseases caninclude neoplastic diseases, such as, melanoma, small cell lung cancer,non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma,thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer,ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrialcancer, kidney cancer, colon cancer, pancreatic cancer, esophagealcarcinoma, head and neck cancers, mesothelioma, sarcomas,cholangiocarcinoma, small bowel adenocarcinoma, pediatric malignanciesand epidermoid carcinoma.

Embodiments of the invention described herein relate to monoclonalantibodies that bind Ang-2 and affect Ang-2 and/or Ang1 function. Otherembodiments relate to fully human anti-Ang-2 antibodies and anti-Ang-2antibody preparations with desirable properties from a therapeuticperspective, including high binding affinity for Ang-2, the ability toneutralize Ang-2 and/or Ang1 in vitro and in vivo, and the ability toinhibit Ang-2 and/or Ang-1 induced angiogenesis.

Another embodiment of the invention is a fully human antibody that bindsto other Angiopoietin-2 family members including, but not limited to,Angiopoietin-1, Angiopoietin-3, and Angiopoietin-4. A further embodimentherein is an antibody that cross-competes for binding to Tie2 with Ang-2with the fully human antibodies of the invention. In one embodiment ofthe invention, the antibody binds to and neutralizes Angiopoietin-2, andalso binds to and neutralizes, Angiopoietin-1.

It will be appreciated that embodiments of the invention are not limitedto any particular form of an antibody or method of generation orproduction. For example, the anti-Ang-2 antibody may be a full-lengthantibody (e.g., having an intact human Fc region) or an antibodyfragment (e.g., a Fab, Fab′ or F(ab′)₂). In addition, the antibody maybe manufactured from a hybridoma that secretes the antibody, or from arecombinantly produced cell that has been transformed or transfectedwith a gene or genes encoding the antibody.

Other embodiments of the invention include isolated nucleic acidmolecules encoding any of the antibodies described herein or portionsthereof, vectors having isolated nucleic acid molecules encodinganti-Ang-2 antibodies or a host cell transformed with any of suchnucleic acid molecules. In addition, one embodiment of the invention isa method of producing an anti-Ang-2 antibody by culturing host cellsunder conditions wherein a nucleic acid molecule is expressed to producethe antibody followed by recovering the antibody. It should be realizedthat embodiments of the invention also include any nucleic acid moleculewhich encodes an antibody or fragment of an antibody of the inventionincluding nucleic acid sequences optimized for increasing yields ofantibodies or fragments thereof when transfected into host cells forantibody production.

Another embodiment of the invention includes a method of diagnosingdiseases or conditions in which an antibody prepared as described hereinis utilized to detect the level of Ang-2 in a patient sample. In oneembodiment, the patient sample is blood or blood serum. In furtherembodiments, methods for the identification of risk factors, diagnosisof disease, and staging of disease is presented which involves theidentification of the overexpression of Ang-2 using anti-Ang-2antibodies.

Another embodiment of the invention includes a method for diagnosing acondition associated with the expression of Ang-2 in a cell bycontacting the serum or a cell with an anti-Ang-2 antibody, andthereafter detecting the presence of Ang-2. Selected conditions includeangiogenesis-related diseases including, but not limited to, neoplasticdiseases, such as, melanoma, small cell lung cancer, non-small cell lungcancer, glioma, hepatocellular (liver) carcinoma, glioblastoma, andcarcinoma of the thyroid, stomach, prostate, breast, ovary, bladder,lung, uterus, kidney, colon, and pancreas, salivary gland, andcolorectum.

In another embodiment, the invention includes an assay kit for detectingAngiopoietin-2 and Angiopoietin family members in mammalian tissues,cells, or body fluids to screen for angiogenesis-related diseases. Thekit includes an antibody that binds to Angiopoietin-2 and a means forindicating the reaction of the antibody with Angiopoietin-2, if present.In one embodiment, the antibody that binds Ang-2 is labeled. In anotherembodiment the antibody is an unlabeled primary antibody and the kitfurther includes a means for detecting the primary antibody. In oneembodiment, the means includes a labeled second antibody that is ananti-immunoglobulin. In other embodiments, the antibody is labeled witha marker selected from the group consisting of a fluorochrome, anenzyme, a radionuclide and a radiopaque material.

Yet another embodiment includes methods for treating diseases orconditions associated with the expression of Ang-2 in a patient, byadministering to the patient an effective amount of an antibody of theinvention. The antibody of the invention can be administered alone, orcan be administered in combination with chemotherapies,biological/immunological therapies, radiation therapies, hormonaltherapies, or surgery. For example, a monoclonal, oligoclonal orpolyclonal mixture of Ang-2 antibodies that block angiogenesis can beadministered in combination with a drug shown to inhibit tumor cellproliferation directly. The method can be performed in vivo and thepatient, in some embodiments, is a human patient. In one embodiment, themethod concerns the treatment of angiogenesis-related diseasesincluding, but not limited to, neoplastic diseases, such as, melanoma,small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, glioblastoma, and carcinoma of thethyroid, stomach, prostate, breast, ovary, bladder, lung, uterus,kidney, colon, and pancreas, salivary gland, and colorectum.

In another embodiment, the invention provides an article of manufactureincluding a container. The container includes a composition containingan antibody of the invention, and a package insert or label indicatingthat the composition can be used to treat angiogenesis-related diseasescharacterized by the overexpression of Ang-2.

In some embodiments, the anti-Ang-2 antibody is administered to apatient, followed by administration of a clearing agent to remove excesscirculating antibody from the blood.

Yet another embodiment is the use of an antibody of the invention in thepreparation of a medicament for the treatment of diseases such asangiogenesis-related diseases. In one embodiment, theangiogenesis-related diseases include carcinoma, such as breast,ovarian, stomach, endometrial, salivary gland, lung, kidney, colon,colorectum, esophageal, thyroid, pancreatic, prostate and bladdercancer. In another embodiment, the angiogenesis-related diseasesinclude, but are not limited to, neoplastic diseases, such as, melanoma,small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, sarcoma, head and neck cancers,mesothelioma, biliary (cholangiocarcinoma), small bowel adenocarcinoma,pediatric malignancies and glioblastoma. In other embodiments,angiogenesis-related disease include, but are not limited tonon-neoplastic diseases, such as psoriasis, arthritis (rheumatoid,osteo, and the like), macular degeneration, restenosis, and others.

Ang-2 is an important “on-switch” of angiogenesis. Accordingly,antagonizing this molecule is expected to inhibit pathophysiologicalprocedures, and thereby act as a potent therapy for variousangiogenesis-dependent diseases. Besides solid tumors and theirmetastases, hematological malignancies, such as leukemias, lymphomas andmultiple myeloma, are also angiogenesis-dependent. Excessive vasculargrowth contributes to numerous non-neoplastic disorders. Thesenon-neoplastic angiogenesis-dependent diseases include: atherosclerosis,hemangioma, hemangioendothelioma, angiofibroma, vascular malformations(e.g. Hereditary Hemorrhagic Teleangiectasia (HHT), or Osler-Webersyndrome), warts, pyogenic granulomas, excessive hair growth, Kaposi'ssarcoma, scar keloids, allergic edema, psoriasis, dysfunctional uterinebleeding, follicular cysts, ovarian hyperstimulation, endometriosis,respiratory distress, ascites, peritoneal sclerosis in dialysispatients, adhesion formation result from abdominal surgery, obesity,rheumatoid arthritis, synovitis, osteomyelitis, pannus growth,osteophyte, hemophilic joints, inflammatory and infectious processes(e.g. hepatitis, pneumonia, glomerulonephritis), asthma, nasal polyps,liver regeneration, pulmonary hypertension, retinopathy of prematurity,diabetic retinopathy, age-related macular degeneration., leukomalacia,neovascular glaucoma, corneal graft neovascularization, trachoma,thyroiditis, thyroid enlargement, and lymphoproliferative disorders.

In other embodiments, the invention provides methods of using antibodiesof the invention in combination with other agents, such asanti-angiogenic or anti-inflammatory agents to treat diseases and/orconditions in a mammal. In one embodiment, methods of the inventioncomprise the combination of anti-Ang-2 antibodies with antagonists ofthe biological activity of Colony Stimulating Factor 1 (CSF1) and/orCSF1 receptor (CSF1R) useful to treat disease.

In other embodiments, the invention provides methods of treatment ofcancer in a patient. More specifically the methods of the invention maycomprise administration of an antagonist of the biological activity ofAngiopoietin-2, and/or Tie-2, in combination with a chemotherapeuticagent; a pharmaceutical composition comprising an antagonist of thebiological activity of Angiopoietin-2, and/or Tie-2, and achemotherapeutic agent; a combination product comprising an antagonistof the biological activity of Angiopoietin-2, and/or Tie-2, and achemotherapeutic agent for use in a method of treatment of a patient; akit comprising an antagonist of the biological activity ofAngiopoietin-2, and/or Tie-2, and a chemotherapeutic agent; to the useof an antagonist of the biological activity of Angiopoietin-2, and/orTie-2, and a chemotherapeutic agent in the manufacture of a medicamentfor use in the production of an anti-cancer effect in a patient. Suchcombinations are also useful for the treatment of other diseasesassociated with the activity of Angiopoietin-2, and/or Tie-2.

IV. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the results from a Differential Scanning Calorimetryexperiment of the wild type antibody 3.19.3 (WT) as well as antibodiescomprising a VL corresponding to MEDI1 and a VH corresponding to MEDI5in both an IgG1 and an IgG2 format. The figure depicts the relativeincrease of melting temperature of the two MEDI1/5 antibodies of the toWT control antibody.

FIG. 2 represents a chromatograph of various preparations of Ang-2antibodies. In (A) the 3.19.3 antibody demonstrates a heterogeneity ofsizes corresponding to various adducts formed with the antibody. (B) and(C) represent the Ang-2 specific antibody MEDI1/5 in the IgG1 (B) andIgG2 (C) formats. These antibodies do not exhibit the heterogeneity insizes displayed by the wild type 3.19.3 antibody.

FIG. 3 represents the results from a competition ELISA based Ang-2binding assay performed on wild type 3.19.3 antibodies as well asMEDI1/5 antibodies in an IgG1 or an IgG2 format. The figure depicts thatthe MEDI1/5 antibodies exhibit a similar binding profile for Ang-2 ascompared to the 3.19.3 antibody as measured by a competition assay withimmobilized Tie-2.

FIG. 4 a demonstrates combination efficacy following treatment with mAb3.19.3 and AZD6495 in mice bearing MCF7 xenograft tumors. The y axisshows the Tumor volume in mm³, against the Days of treatment, in whichthe square points represent vehicle; circular points represent mAb3.19.3; triangular points represent AZD6495; diamond points representmAb 3.19.3 and AZD6495 combination.

FIG. 4 b demonstrates effects on host body weight changes followingcombination treatment with mAb 3.19.3 and AZD6495 in mice bearing MCF7xenograft tumors. The y axis shows the Body weight in g, against theDays of treatment, in which the square points represent vehicle;circular points represent mAb 3.19.3; triangular points representAZD6495; diamond points represent mAb 3.19.3 and AZD6495 combination.

FIG. 5 a demonstrates combination efficacy following treatment with mAb3.19.3 and AZD6495 in mice bearing MDA-MB-231 xenograft tumors. The yaxis shows the Tumor volume in mm³, against the Days of treatment, inwhich the square points represent vehicle; circular points represent mAb3.19.3; triangular points represent AZD6495; diamond points representmAb 3.19.3 and AZD6495 combination.

FIG. 5 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and AZD6495 in mice bearingMDA-MB-231 xenograft tumors. The y axis shows the Body weight in g,against the Days of treatment, in which the square points representvehicle; circular points represent mAb 3.19.3; triangular pointsrepresent AZD6495; diamond points represent mAb 3.19.3 and AZD6495combination.

FIG. 6 a. Shows combination efficacy following treatment with mAb 3.19.3and 5-flurouracil in mice bearing LoVo xenograft tumors.

FIG. 6 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and 5-fluoruracil in mice bearingLoVo xenograft tumors.

FIG. 7 a. Shows combination efficacy following treatment with mAb 3.19.3and Irinotecan in mice bearing HT-29 xenograft tumors.

FIG. 7 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and Irinotecan in mice bearingHT29 xenograft tumors.

FIG. 8 a. Shows combination efficacy following treatment with mAb 3.19.3and Gemcitabine in mice bearing Colo205 xenograft tumors.

FIG. 8 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and Gemcitabine in mice bearingColo205 xenograft tumors.

FIG. 9 a. Shows combination efficacy following treatment with mAb 3.19.3and Docetaxel in mice bearing Calu6 xenograft tumors.

FIG. 9 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and Docetaxel in mice bearingCalu6 xenograft tumors.

FIG. 10 a. Shows combination efficacy following treatment with mAb3.19.3 and Oxaliplatin in mice bearing H460 xenograft tumors.

FIG. 10 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and Oxaliplatin in mice bearingH460 xenograft tumors.

FIG. 11 a. Shows combination efficacy following treatment with mAb3.19.3 and AZD4877 in mice bearing H460 xenograft tumors.

FIG. 11 b. Shows effects on host body weight changes followingcombination treatment with mAb 3.19.3 and AZD4877 in mice bearing H460xenograft tumors.

FIG. 12 a. Effect of 3.19.3 treatment on clinical disease progression inthe collagen induced arthritis disease model. Collagen induced arthritiswas induced in male DBA/1 mice and animals dosed therapeutically withtest treatments. FIG. 12 a shows the Arthritic score mean (+/− standarderror of the mean) against Days from disease onset (i.e. Days oftreatment). Closed squares represent PBS vehicle treated animals (n=15),Open triangles represent human IgG isotype control treated animals(n=15), closed circles represent 3.19.3 10 mg/kg treated animals (n=15)and open squares represent Prednisolone 3 mg/kg treated animals (n=10).

FIG. 12 b: Effect on mean animal body weights. FIG. 12 b shows that nosignificant changes in mean body weight (g) were observed between eachtreatment group throughout time course of collagen induced arthritis,suggesting the 3.19.3 therapy was well tolerated. FIG. 12 b shows Bodyweight in grams against Days from disease onset (i.e. Days oftreatment). Closed squares represent PBS vehicle treated animals (n=15),Open triangles represent human IgG isotype control treated animals(n=15), closed circles represent 3.19.3 10 mg/kg treated animals (n=15)and open squares represent Prednisolone 3 mg/kg treated animals (n=10).

FIG. 13: Anti-Ang-2 antibodies inhibit retinal angiogenesis. FIG. 13represents alterations in angiogenesis of murine retinas in control pups(a) and in pups treated with 0.3 mg/kg MEDI1/5 (b), 1.0 mg/kg MEDI1/5(c), and 10 mg/kg MEDI1/5 (d). These panels demonstrate that murineretinal angiogenesis is inhibited in a dose-dependent fashion withMEDI1/5 anti-Ang-2 antibodies, as compared to animals treated withcontrol antibodies.

FIG. 14. Anti-Ang-2 antibodies inhibit FGF2 mediated angiogenesis. FIG.14 represents results demonstrating the inhibition of FGF2 mediatedangiogenesis in mice by the administration of the anti-Ang-2 antibody,MEDI1/5. Briefly, Matrigel™ was mixed with FGF2 and implantedsubcutaneously into athymic nude mice. MEDI1/5 was dosedintraperitoneally at 1, 10 or 20 mg/kg, on days 1, 4 and 8 of implant.On day 11 post-implant, mice were intravenously dosed with FITC-dextranand Matrigel™ plugs were harvested. Plugs were quantitated forFITC-Dextran content (a) all three doses of MEDI1/5 resulted insignificant reduction in angiogenesis (*p<0.05). Plugs were alsoprepared hematoxylin and eosin staining (b) which showed a lower levelof vascularization as compared to the control FGF2 treated sample.

FIG. 15: Anti-Ang-2 antibodies inhibit arthritis disease progression.FIG. 15A represents the global arthritic score of arthritis-inducedanimals treated with various agents including the anti-Ang-2 antibody,3.19.3 and Prednisolone (open squares=PBS, open triangles=isotypecontrol, closed squares=0.1 mg/kg 3.19.3, closed triangles=1 mg/kg3.19.3, closed circles=10 mg/kg 3.19.3 and open circles=prednisolone).Dose-dependent reductions in clinical signs of disease progression(arthritic score) and were observed. There was a significant reductionat doses of 1 and 10 mg/kg of 3.19.3. Area under the curve (AUC) forclinical disease progression was calculated for each animal from diseaseonset and presented in FIG. 15B. FIGS. 15C-H further demonstrate theameliorative effect of treatment with the anti-Ang-2 antibody 3.19.3.Histolopathological evaluation of CIA model showed evidence of adose-dependent anti-arthritic effect following administration of 3.19.3on all parameters evaluated including synovial hyperplasmia (FIG. 15 C),synovitis (FIG. 15 D), pannus (FIG. 15 E), synovial fibrosis (FIG. 15F),and periostitis (FIG. 15G). Histologically, there were no significantdifferences between the isotype control-treated group and the PBSvehicle group (FIGS. 15C-G). Further, investigation into the microvesseldensity using CD31 staining showed a significant reduction in thepresence of microvessels in the synovium at doses of 1 and 10 mg/kg aswell as with prednisolone. There was no effect with 0.1 mg/kg 3.19.3treatment (FIG. 15 H).

FIG. 16: Combinations of Anti-Ang-2 and Anti-TNFα agents demonstrateefficacy in prophylactic treatment of arthritis. FIGS. 16A+B representthe arthritic score of arthritis-induced animals prophylacticallytreated with a combination of MEDI1/5+ etanercept (closedcircles=isotype control, closed diamond=10 mg/kg MEDI1/5, open diamond=1mg/kg etanercept, grey diamond=combination of 10 mg/kg MEDI1/5 with 1mg/kg etanercept, open square=4 mg/kg enbrel, grey square=combination of10 mg/kg MEDI1/5 with 4 mg/kg etanercept). A reduction was observed witheither etanercept or MEDI1/5 treatment. There was a further reduction inclincal score when MEDI1/5 was administered in combination with thelower dose of etanercept. Histological assessment of synovitis and jointdestruction (FIG. 16C) supported the clinical score results as did theprotection from loss of bone mineral density (FIG. 16D).

FIG. 17: Combinations of Anti-Ang-2 and Anti-TNFα agents demonstrateefficacy in therapeutic treatment of arthritis When administered in atherpeutic approach following the onset of clinical disease, modestreductions in clinical signs of disease progression (arthritic score)were observed with MEDI1/5 treatment, while both doses of etanercepttested had no effect on disease progression. A more dramatic inhibitionof progression of disease when MEDI1/5 (10 mg/kg) was administered incombination with the higher dose of etanercept (4 mg/kg) (FIG. 17(closed circles=isotype control, closed diamond=MEDI1/5,open diamond=1mg/kg etanercept, grey diamond=combination of MEDI1/5 with 1 mg/kgetanercept, open square=4 mg/kg etanercept, grey square=combination ofMEDI1/5 with 4 mg/kg etanercept).

V. DETAILED DESCRIPTION

The inventors have found that certain modifications could be made to aparticular Ang-2 antibody, which renders the antibody more stable undercertain conditions. In particular, by altering residue 49 of the lightchain much less aggregation occurred. In addition, when residue 37 ofthe heavy chain was changed, much less aggregation occurred.

Accordingly, in one embodiment the invention is directed to Ang-2antibodies having one or more improved characteristics over controlantibody 3.19.3. Such characteristics include increased stability,decreased aggregation and increased production efficiency. In oneembodiment, the antibodies of the invention efficiently inhibit Ang-2and/or Ang-1 signaling through the Tie2 receptor to modulate processessuch as angiogenesis and tumor growth.

Embodiments of the invention described herein relate to monoclonalantibodies specific for Ang-2, which may be derived from the antibody3.19.3 and which exhibit increased stability and/or productionefficiencies. In some embodiments, the antibodies bind to Ang-2 andinhibit the binding of Ang-2 to its receptor, Tie2. Other embodiments ofthe invention include fully human anti-Ang-2 antibodies, and antibodypreparations that are therapeutically useful. Such anti-Ang-2 antibodypreparations have desirable therapeutic properties, including strongbinding affinity for Ang-2, the ability to neutralize Ang-2 in vitro,and the ability to inhibit Ang-2-induced angiogenesis in vivo.Antibodies of the invention comprise the ability to specifically bindAng-2 and inhibit tumor angiogenesis and reduce tumor growth. Mechanismsby which this can be achieved can include and are not limited to eitherinhibition of binding of Ang-2 to its receptor Tie2, inhibition of Ang-2induced Tie2 signaling, or increased clearance of Ang-2, thereinreducing the effective concentration of Ang-2.

In other embodiments, the antibodies may bind to both Ang-2 and Ang-1and/or modulate one or more functional activities of both Ang-1 andAng-2.

One aspect of the invention provides stabilized antibodies whichcomprise a substitution of an amino acid at position 49 (as compared tothe light chain variable amino acid sequence of Ang-2 antibody 3.19.3,see SEQ ID No.1) as defined by the Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NIHPublication 91-3242, Bethesda Md. (1991), vols. 1-3.). In oneembodiment, the amino acid substitution at position 49 may be any aminoacid. In a specific embodiment, the amino acid substitution at position49 is selected from the group consisting of Asp, Thr, Asn, and Ala.

In another embodiment, antibodies of the invention further comprises asubstitution of Val substitution at position 37 of the heavy chain (ascompared to the heavy chain variable amino acid sequence of Ang-2antibody 3.19.3, see, SEQ ID No. 2) as defined by the EU numberingsystem ((Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols.1-3.).

In one embodiment, the antibodies of the invention exhibit enhancedstability as compared to antibody 3.19.3. In another embodiment, theantibodies of the invention exhibit enhanced production yields ascompared to 3.19.3.

In another embodiment, the antibodies of the invention may comprisevariable light chain acid sequences selected from the group consistingof MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID No.:5),MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6). In another embodiment,the antibodies of the invention may comprise the heavy chain variablesequence of MEDI5 (SEQ ID No.:7). In another embodiment, the antibodiesof the invention may comprise variable light chain sequences selectedfrom the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6) andfurther comprise the heavy chain variable sequence of MEDI5 (SEQ IDNo.:7). As used herein, an antibody of the invention comprising a lightchain and a heavy chain may be referred to as a MEDIX/MEDIY wherein Xrepresents the light chain sequence and Y represents the heavy chainsequence.

In another embodiment, the antibodies of the invention may comprisevariable light chain acid sequences selected from the group consistingof MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID No.:5),MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6), but having a differentamino acid substitution at position 49. In another embodiment, theantibodies of the invention may further comprise the heavy chainvariable sequence of MEDI5 (SEQ ID No.:7). In another embodiment, theantibodies of the invention may comprise variable light chain sequencesselected from the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQID No.:4), MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ IDNo.:6), but having a different amino acid substitution at position 49,and further comprise the heavy chain variable sequence of MEDI5 (SEQ IDNo.:7).

Modulation of Unpaired Cysteine Residues:

Disulfide bond formation in proteins is a complex process, which isdetermined by the redox potential of the environment and specializedthiol-disulfide exchanging enzymes (Creighton, Methods Enzymol. 107,305-329, 1984; Houee-Levin, Methods Enzymol. 353, 35-44,2002),Ingeneral, cysteine residues in proteins (for example, antibodies specificfor Ang-2) are either engaged in cysteine-cysteine disulfide bonds orsterically protected from the disulfide bond formation when they are apart of folded protein region. When a cysteine residue does not have apair in protein structure and is not sterically protected by folding, itcan form a disulfide bond with a free cysteine from solution in aprocess known as disulfide shuffling. In another process known asdisulfide scrambling, free cysteines may also interfere with naturallyoccurring disulfide bonds (such as those present in antibody structures)and lead to low binding, low biological activity and/or low stability.

Modulation of Glycosylation Sites:

It has been shown that glycosylation in the variable region can have anegative effect on antibody binding affinity, likely due to sterichindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367). Theheterogeneity of the glycosylation process may also lead to a number ofantibody species with altered binding properties. As such, it isdesirable to remove or alter the interfering glycosylation site toensure a consistent antigen binding profile. One method to removepotential or observed glycosylation sites is site-directed mutagenesisto substitute at least one potential glycosylation site (such as anasparagine, threonine or serine amino acid) with another amino acid thatcannot serve as a glycosylation site. Accordingly, in one embodiment,the antibodies of the invention comprise substituted amino acids that donot serve as glycosylation sites. In one embodiment, the glycosylationsite to be modified occurs in the variable region. In anotherembodiment, the glycosylation site to be modified occurs in acomplementary determining region (CDR) of an antibody. In anotherembodiment, the glycosylation site to be modified is the 2^(nd) lightchain CDR. In other embodiments, the sequences surrounding theglycosylation site are modified. In another embodiment, theglycosylation site to be modified occurs in the constant region. Inanother embodiment, the antibodies of the invention comprise at leastone, at least two, at least three, at least four or more modifiedglycosylation sites.

In some embodiments, antibodies of the invention comprise a light chainwhich is engineered to remove at least one O-glycosylation site. In someembodiments, antibodies of the invention comprise a light chain selectedfrom the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),MEDI3 (SEQ ID No.:5), and MEDI4 (SEQ ID No.:6) wherein said light chainfurther comprises an amino acid substitution at Kabat position 59,wherein said amino acid is not proline. In a specific embodiment,antibodies of the invention comprise a light chain having the sequencecorresponding to MEDI6 (SEQ ID NO:8).

The amino acid sequences of selected antibody heavy and light chains canbe compared to germline heavy and light chain amino acid sequences. Incases where certain framework residues of the selected VL and/or VHchains differ from the germline configuration (e.g., as a result ofsomatic mutation of the immunoglobulin genes used to prepare the phagelibrary), it may be desirable to “backmutate” the altered frameworkresidues of the selected antibodies to the germline configuration (i.e.,change the framework amino acid sequences of the selected antibodies sothat they are the same as the germline framework amino acid sequences).Such “backmutation” (or “germlining”) of framework residues can beaccomplished by standard molecular biology methods for introducingspecific mutations (e.g., site-directed mutagenesis; PCR-mediatedmutagenesis, and the like). In one embodiment, the variable light and/orheavy chain framework residues are backmutated. In another embodiment,the variable heavy chain of an antibody of the invention is backmutated.In another embodiment, the variable heavy chain of an antibody of theinvention comprises at least one, at least two, at least three, at leastfour or more backmutations. In a specific embodiment, the variable heavychain of an antibody of the invention comprises a backmutation of theglycine residue occupying position 37. In another specific embodiment,the variable heavy chain of an antibody of the invention comprises abackmutation of position 37 corresponding to a glycine to valinesubstitution.

Modulation of the Fc Region

The invention also provides antibodies with altered Fc regions (alsoreferred to herein as “variant Fc regions”). Accordingly, in oneembodiment of the invention, antibodies of the invention comprise avariant Fc region (i.e., Fc regions that have been altered as discussedbelow). Antibodies of the invention comprising a variant Fc region arealso referred to here as “Fc variant protein(s).”

In the description of variant Fc regions, it is understood that the Fcregions of the antibodies of the invention comprise the numbering schemeaccording to the EU index as in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.).

It is known that variants of the Fc region (e.g., amino acidsubstitutions and/or additions and/or deletions) enhance or diminisheffector function (see Presta et al., 2002, Biochem Soc Trans30:487-490; U.S. Pat. Nos. 5,624,821, 5,885,573 and PCT publication Nos.WO 00/42072, WO 99/58572 and WO 04/029207). Accordingly, in oneembodiment, the antibodies of the invention comprise variant Fc regions.In one embodiment, the variant Fc regions of antibodies exhibit asimilar level of inducing effector function as compared to the nativeFc. In another embodiment, the variant Fc region exhibits a higherinduction of effector function as compared to the native Fc. In anotherembodiment, the variant Fc region exhibits lower induction of effectorfunction as compared to the native Fc. In another embodiment, thevariant Fc region exhibits higher induction of ADCC as compared to thenative Fc. In another embodiment, the variant Fc region exhibits lowerinduction of ADCC as compared to the native Fc. In another embodiment,the variant Fc region exhibits higher induction of CDC as compared tothe native Fc. In another embodiment, the variant Fc region exhibitslower induction of CDC as compared to the native Fc. Specificembodiments of variant Fc regions are detailed infra.

It is also known in the art that the glycosylation of the Fc region canbe modified to increase or decrease effector function (see for examples,Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.);GlycoMAb™ glycosylation engineering technology (GLYCART biotechnologyAG, Zurich, Switzerland). Accordingly, in one embodiment the Fc regionsof antibodies of the invention comprise altered glycosylation of aminoacid residues. In another embodiment, the altered glycosylation of theamino acid residues results in lowered effector function. In anotherembodiment, the altered glycosylation of the amino acid residues resultsin increased effector function. In a specific embodiment, the Fc regionhas reduced fucosylation. In another embodiment, the Fc region isafucosylated (see for examples, U.S. Patent Application Publication No.2005/0226867).

Recent research suggests that the addition of sialic acid to theoligosaccharides on IgG molecules enhances their anti-inflammatoryactivity and alter their cytotoxicity (Keneko et al., Science 313,670-673(2006), Scallon et al., Mol. Immuno. 2007 March; 44(7):1524-34).Thus, the efficacy of antibody therapeutics may be optimized byselection of a glycoform that is best suited to the intendedapplication. The two oligosaccharide chains interposed between the twoCH2 domains of antibodies are involved in the binding of the Fc regionto its receptors. The studies referenced above demonstrate that IgGmolecules with increased sialylation have anti-inflammatory propertieswhereas IgG molecules with reduced sialylation have increasedimmunostimulatory properties. Therefore, an antibody therapeutic can be“tailor-made” with an appropriate sialylation profile for a particularapplication. Methods for modulating the sialylation state of antibodiesare presented in WO2007/005786 entitled “Methods And Compositions WithEnhanced Therapeutic Activity”, and WO2007/117505 entitled “PolypeptidesWith Enhanced Anti-Inflammatory And Decreased Cytotoxic Properties AndRelated Methods” each of which are incorporated by reference in theirentireties for all purposes.

In one embodiment, the Fc regions of antibodies of the inventioncomprise an altered sialylation profile compared to a referenceunaltered Fc region. In one embodiment, the Fc regions of antibodies ofthe invention comprise an increased sialylation profile compared to areference unaltered Fc region. In some embodiments the Fc regions ofantibodies of the invention comprise an increase in sialylation of about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%or more as compared to a reference unaltered Fc region. In someembodiments the Fc regions of antibodies of the invention comprise anincrease in sialylation of about 2 fold, about 3 fold, about 4 fold,about 5 fold, about 10 fold, about 20 fold, about 50 fold or more ascompared to an unaltered reference Fc region.

In another embodiment, the Fc regions of antibodies of the inventioncomprise a decreased sialylation profile compared to a referenceunaltered Fc region. In some embodiments, the Fc regions of antibodiesof the invention comprise a decrease in sialylation of about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about85%, about 90%, about 95%, about 100%, about 125%, about 150% or more ascompared to a reference unaltered Fc region. In some embodiments the Fcregions of antibodies of the invention comprise a decrease insialylation of about 2 fold, about 3 fold, about 4 fold, about 5 fold,about 10 fold, about 20 fold, about 50 fold or more as compared to anunaltered reference Fc region.

It is also known in the art that the Fc region can be modified toincrease the half-lives of proteins. The increase in half-life allowsfor the reduction in amount of drug given to a patient as well asreducing the frequency of administration. Accordingly, antibodies of theinvention with increased half-lives may be generated by modifying (forexample, substituting, deleting, or adding) amino acid residuesidentified as involved in the interaction between the Fc and the FcRnreceptor (see, for examples, PCT publication Nos. 97/34631 and 02/060919each of which are incorporated by reference in their entireties). Inaddition, the half-life of antibodies of the invention may be increaseby conjugation to PEG or Albumin by techniques widely utilized in theart. In some embodiments the Fc regions of antibodies of the inventioncomprise an increase in half-life of about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%,about 95%, about 100%, about 125%, about 150% or more as compared to areference unaltered Fc region. In some embodiments the Fc regions ofantibodies of the invention comprise an increase in half-life of about 2fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20fold, about 50 fold or more as compared to an unaltered reference Fcregion.

The present invention encompasses Fc variant proteins which have alteredbinding properties for an Fc ligand (e.g., an Fc receptor, C1q) relativeto a comparable molecule (e.g., a protein having the same amino acidsequence except having a wild type Fc region). Examples of bindingproperties include but are not limited to, binding specificity,equilibrium dissociation constant (K_(D)), dissociation and associationrates (k_(off) and k_(on) respectively), binding affinity and/oravidity. It is generally understood that a binding molecule (e.g., a Fcvariant protein such as an antibody) with a low K_(D) may be moredesirable to a binding molecule with a high K_(D). However, in someinstances the value of the k_(on) or k_(off) may be more relevant thanthe value of the K_(D). One skilled in the art can determine whichkinetic parameter is most important for a given antibody application.

The affinities and binding properties of an Fc region for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In one embodiment, the Fc variant protein has enhanced binding to one ormore Fc ligand relative to a comparable molecule. In another embodiment,the Fc variant protein has an affinity for an Fc ligand that is at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold greater than that of a comparable molecule. In a specificembodiment, the Fc variant protein has enhanced binding to an Fcreceptor. In another specific embodiment, the Fc variant protein hasenhanced binding to the Fc receptor FcγRIIIA. In a further specificembodiment, the Fc variant protein has enhanced biding to the Fcreceptor FcγRIIB. In still another specific embodiment, the Fc variantprotein has enhanced binding to the Fc receptor FcRn. In yet anotherspecific embodiment, the Fc variant protein has enhanced binding to C1qrelative to a comparable molecule.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. In oneembodiment, the Fc variant protein has enhanced serum half life relativeto comparable molecule.

The ability of any particular Fc variant protein to mediate lysis of thetarget cell by ADCC can be assayed. To assess ADCC activity an Fcvariant protein of interest is added to target cells in combination withimmune effector cells, which may be activated by the antigen antibodycomplexes resulting in cytolysis of the target cell. Cytolysis isgenerally detected by the release of label (e.g. radioactive substrates,fluorescent dyes or natural intracellular proteins) from the lysedcells. Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361;Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al.,1995 J Immunol Methods 184:29-38. ADCC activity of the Fc variantprotein of interest may also be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad.Sci. USA 95:652-656.

In one embodiment, an Fc variant protein has enhanced ADCC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has ADCC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold greater than that of a comparable molecule. In anotherspecific embodiment, an Fc variant protein has enhanced binding to theFc receptor FcγRIIIA and has enhanced ADCC activity relative to acomparable molecule. In other embodiments, the Fc variant protein hasboth enhanced ADCC activity and enhanced serum half life relative to acomparable molecule.

In one embodiment, an Fc variant protein has reduced ADCC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has ADCC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold lower than that of a comparable molecule. In anotherspecific embodiment, an Fc variant protein has reduced binding to the Fcreceptor FcγRIIIA and has reduced ADCC activity relative to a comparablemolecule. In other embodiments, the Fc variant protein has both reducedADCC activity and enhanced serum half life relative to a comparablemolecule.

In one embodiment, an Fc variant protein has enhanced CDC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has CDC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold greater than that of a comparable molecule. In otherembodiments, the Fc variant protein has both enhanced CDC activity andenhanced serum half life relative to a comparable molecule. In oneembodiment, the Fc variant protein has reduced binding to one or more Fcligand relative to a comparable molecule. In another embodiment, the Fcvariant protein has an affinity for an Fc ligand that is at least 2fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold lower than that of a comparable molecule. In a specificembodiment, the Fc variant protein has reduced binding to an Fcreceptor. In another specific embodiment, the Fc variant protein hasreduced binding to the Fc receptor FcγRIIIA. In a further specificembodiment, an Fc variant described herein has an affinity for the Fcreceptor FcγRIIIA that is at least about 5 fold lower than that of acomparable molecule, wherein said Fc variant has an affinity for the Fcreceptor FcγRIIB that is within about 2 fold of that of a comparablemolecule. In still another specific embodiment, the Fc variant proteinhas reduced binding to the Fc receptor FcRn. In yet another specificembodiment, the Fc variant protein has reduced binding to C1q relativeto a comparable molecule.

In one embodiment, the present invention provides Fc variants, whereinthe Fc region comprises a non naturally occurring amino acid residue atone or more positions selected from the group consisting of 234, 235,236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255,256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443as numbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non naturally occurring amino acid residue atadditional and/or alternative positions known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid residue selected from the group consisting of 234D, 234E,234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W,235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D,239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W,241 L, 241Y, 241E, 241 R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L,247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E,263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y,264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I,266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A,284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H,269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S,299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E,325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E,328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K,330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A,331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y,331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q,332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H,421K, 440Y and 434W as numbered by the EU index as set forth in Kabat.Optionally, the Fc region may comprise additional and/or alternative nonnaturally occurring amino acid residues known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752 and WO 05/040217).

Antibody Affinity

In one embodiment of the invention there is provided an antibody thatbinds to Angiopoietin-1 and prevents Angiopoietin-1 binding to Tie-2.Yet another embodiment of the invention is a monoclonal antibody thatbinds to Angiopoietin-1 and/or Angiopoietin-2 and inhibitsAngiopoietin-1 and/or Angiopoietin-2 induced Tie-2 phosphorylation. Inone embodiment, the antibody binds Angiopoietin-1 and/or Angiopoietin-2with a K_(d) of less than 1 nanomolar (nM). In other embodiments, theantibody binds with a K_(d) less than 500 picomolar (pM). In otherembodiments, the antibody binds with a K_(d) less than 100 picomolar(pM). In yet other embodiments, the antibody binds with a K_(d) lessthan 30 picomolar (pM). In further embodiments, the antibody binds witha K_(d) of less than 20 pM. In yet further embodiments, the antibodybinds with a K_(d) of less than 10 or 5 pM.

Antibodies of the invention may have a high binding affinity Ang-1and/or Ang-2. For example, an antibody described herein may have anassociation rate constant or k_(on) rate (antibody (Ab)+antigen->Ab-Ag)of at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s10⁶ M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹.

In another embodiment, an antibody may have a k_(off) rate (Ab-Ag->Ab+Ag) of less than 5×10⁻¹s⁻¹, less than 10⁻¹s⁻¹, less than 5×10⁻²s⁻¹, less than 10⁻² s⁻¹ , less than 5×10⁻³ s⁻¹, less than 10⁻³ s⁻¹, lessthan 5×10⁻⁴ s⁻¹, or less than 10⁻⁴ s⁻¹. In a another embodiment, anantibody of the invention has a k_(off) of less than 5×10⁻⁵ s⁻¹, lessthan 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁶ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁸s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, an antibody may have an affinity constant orK_(a) (k_(on)/k_(off)) of at least 10² M⁻¹, at least 5×10² M⁻¹, at least10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, atleast 10⁵ M⁻¹, at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹,at least 10⁷ M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸M⁻¹, at least 10⁹ M⁻¹, at least 5 ×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹,at least 5×10¹² M⁻¹, at least 10¹³M⁻¹, at least 5×10¹³ M⁻¹, at least10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵M⁻¹. In yet another embodiment, an antibody may have a dissociationconstant or K_(d) (k_(off)/k_(on)) of less than 5×10⁻² M, less than 10⁻²M, less than 5×10⁻³ M, less than 10⁻³ M, less than 5×10^(—4) M, lessthan 10⁻⁴ M, less than 5×10⁻⁵ M, less than 10⁻⁵ M, less than 5×10⁻⁶ M,less than 10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸ M, less than 5×10⁻⁹ M, less than 10⁻⁹ M, less than5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than 5×10⁻¹¹ M, less than 10⁻¹¹ M,less than 5×10⁻¹² M, less than 10⁻¹² M, less than 5×10⁻¹³M, less than10⁻¹³ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁵ M, orless than 10⁻¹⁵ M.

An antibody used in accordance with a method described herein may have adissociation constant (K_(d)) of less than 3000 pM, less than 2500 pM,less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150pM, less than 100 pM, less than 75 pM as assessed using a methoddescribed herein or known to one of skill in the art (e.g., a BlAcoreassay, ELISA) (Biacore International AB, Uppsala, Sweden). In a specificembodiment, an antibody used in accordance with a method describedherein may have a dissociation constant (K_(d)) of between 25 to 3400pM, 25 to 3000 pM, 25 to 2500 pM, 25 to 2000 pM, 25 to 1500 pM, 25 to1000 pM, 25 to 750 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, 25 to75 pM, or 25 to 50 pM as assessed using a method described herein orknown to one of skill in the art (e.g., a BlAcore assay, ELISA). Inanother embodiment, an antibody used in accordance with a methoddescribed herein may have a dissociation constant (K_(d)) of 500 pM, 100pM, 75 pM or 50 pM as assessed using a method described herein or knownto one of skill in the art (e.g., a BlAcore assay, ELISA).

One embodiment of the invention includes an antibody that binds to andneutralizes Ang-2, but does not bind to Ang-1. In another embodiment,the antibody binds to both Ang-2 and Ang-1, but only neutralizes Ang-2.In another embodiment, the antibody binds to both Ang-2 and Ang-1, andneutralizes binding of both Ang-1 and Ang-2 to Tie2.

In one embodiment, antibodies of the invention preferentially bind Ang-2over Ang-1. In some embodiments, antibodies of the invention bind Ang-2over Ang-1 in a ratio of at least 2:1, at least 3:1, at least 4:1, atleast 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, atleast 10:1, at least 15:1. at least 20:1, at least 25:1, at least 50:1,at least 100:1, at least 250:1, at least 500:1, at least 1000:1 or atleast 10,000:1 or higher.

In one embodiment, antibodies of the invention preferentially bind Ang-1over Ang-2. In some embodiments, antibodies of the invention bind Ang-1over Ang-2 in a ratio of at least 2:1, at least 3:1, at least 4:1, atleast 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, atleast 10:1, at least 15:1. at least 20:1, at least 25:1, at least 50:1,at least 100:1, at least 250:1, at least 500:1, at least 1000:1 or atleast 10,000:1 or higher.

Embodiments of the invention also include isolated binding fragments ofanti-Ang-2 antibodies. In one embodiment, the binding fragments arederived from fully human anti-Ang-2 antibodies. Exemplary fragmentsinclude Fv, Fab' or other well know antibody fragments, as described inmore detail below. Embodiments of the invention also include cells thatexpress fully human antibodies against Ang-2. Examples of cells includehybridomas, or recombinantly created cells, such as Chinese hamsterovary (CHO) cells, variants of CHO cells (for example DG44), 293 cellsand NSO cells that produce antibodies against Ang-2. Additionalinformation about variants of CHO cells can be found in Andersen andReilly (2004) Current Opinion in Biotechnology 15, 456-462 which isincorporated herein in its entirety by reference.

Preparation of Antibodies Nucleic Acids Encoding Antibodies of theInvention

The invention also encompasses isolated nucleic acid molecules encodingantibodies of the invention. In another embodiment, the antibody isderived from the fully human monoclonal antibody 3.19.3. In oneembodiment there is provided an antibody which binds to the same epitopeor epitopes as fully human monoclonal antibody 3.19.3.

In one embodiment, the isolated nucleic acid encodes an antibodyvariable light chain corresponding to an amino acid sequence selectedfrom the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6). Inanother embodiment, the isolated nucleic acid encodes an antibodyfurther comprising a variable heavy chain corresponding to the aminoacid sequence MEDI5 (SEQ ID NO:7). In a specific embodiment, the nucleicacids of the invention encode an antibody comprising a variable lightchain corresponding to an amino acid sequence selected from the groupconsisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ IDNo.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6); and furthercomprises a variable heavy chain further comprising the amino acidsequence MEDI5 (SEQ ID No.: 7).

Recombinant Expression Systems

Recombinant expression of an antibody of the invention requiresconstruction of an expression vector containing a polynucleotide thatencodes the antibody of the invention. Once a polynucleotide encodingthe antibody of the invention has been obtained, the vector for theproduction of the antibody may be produced by recombinant DNA technologyusing techniques well-known in the art (e.g., U.S. Pat. No. 6,331,415,which is incorporated herein by reference in its entirety). Thus,methods for preparing a protein by expressing a polynucleotidecontaining an encoding nucleotide sequence are described herein. Theantibodies of the invention can be produced in many different expressionsystems. In one embodiment, the antibodies of the invention are producedand secreted by mammalian cells. In another embodiment, the antibodiesof the invention are produced and secreted in human cells. In a specificembodiment, the antibodies of the invention are produced in cells of the293F, CHO, or NSO cell line.

Methods which are known to those skilled in the art can be used toconstruct expression vectors containing protein coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule operably linked to a promoter.

Once the expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding a protein of the inventionoperably linked to a heterologous promoter.

A variety of host-expression vector systems may be utilized to expressantibodies of the invention or portions thereof as described in U.S.Pat. No. 5,807,715. For example, mammalian cells such as Chinese hamsterovary cells (CHO), in conjunction with a vector such as the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for antibodies (Foecking et al., Gene,45:101 (1986); and Cockett et al., Bio/Technology, 8:2 (1990)). Inaddition, a host cell strain may be chosen which modulates theexpression of inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the protein of the invention. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293F, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO,CRL7O3O and HsS78Bst cells.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinmolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions comprising an antibody of the invention, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278 (Ruther et al., EMBO,12:1791 (1983)), in which the coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J.Biol. Chem., 24:5503-5509 (1989)); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione-S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to glutathione-agarose affinity matrix followed by elution inthe presence of free glutathione. The pGEX vectors are designed tointroduce a thrombin and/or factor Xa protease cleavage sites into theexpressed polypeptide so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The protein coding sequence may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter).

In mammalian host cells, a number of virus based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see, Logan &Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon should generally be in frame with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, e.g., Bittneret al., Methods in Enzymol., 153:51-544(1987)).

Stable expression can be used for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express theprotein molecule may be generated. Host cells can be transformed with anappropriately engineered vector comprising expression control elements(e.g., promoter, enhancer, transcription terminators, polyadenylationsites, etc.), and a selectable marker gene. Following the introductionof the foreign DNA, cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells that stably integrated the plasmid into theirchromosomes to grow and form foci which in turn can be cloned andexpanded into cell lines. Plasmids that encode an antibody of theinvention can be used to introduce the gene/cDNA into any cell linesuitable for production in culture.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell,11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes canbe employed in tk-, hgprt- or aprT-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH11(5):155-2 15 (1993)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981,J. Mol. Biol., 150:1, which are incorporated by reference herein intheir entireties.

Once an antibody of the invention has been produced by recombinantexpression, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigens Protein A or Protein G, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theproteins of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

Scalable Production of Antibodies

In an effort to obtain large quantities, antibodies of the invention maybe produced by a scalable process (hereinafter referred to as “scalableprocess of the invention”). In some embodiments, antibodies may beproduced by a scalable process of the invention in the researchlaboratory that may be scaled up to produce the antibodies of theinvention in analytical scale bioreactors (for example, but not limitedto 5L, 10L, 15L, 30L, or 50L bioreactors). In other embodiments, theantibodies may be produced by a scalable process of the invention in theresearch laboratory that may be scaled up to produce the antibodies ofthe invention in production scale bioreactors (for example, but notlimited to 75L, 100L, 150L, 300L, or 500L). In some embodiments, thescalable process of the invention results in little or no reduction inproduction efficiency as compared to the production process performed inthe research laboratory. In other embodiments, the scalable process ofthe invention produces antibodies at production efficiency of about 10mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L, about100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200mg/L, about 250 mg/L, or about 300 mg/L or higher.

In other embodiments, the scalable process of the invention producesantibodies at production efficiency of at least about 10 mg/L, at leastabout 20 m/L, at least about 30 mg/L, at least about 50 mg/L, at leastabout 75 mg/L, at least about 100 mg/L, at least about 125 mg/L, atleast about 150 mg/L, at least about 175 mg/L, at least about 200 mg/L,at least about 250 mg/L, or at least about 300 mg/L or higher.

In other embodiments, the scalable process of the invention producesantibodies at production efficiency from about 10 mg/L to about 300mg/L, from about 10 mg/L to about 250 mg/L, from about 10 mg/L to about200 mg/L, from about 10 mg/L to about 175 mg/L, from about 10 mg/L toabout 150 mg/L, from about 10 mg/L to about 100 mg/L, from about 20 mg/Lto about 300 mg/L, from about 20 mg/L to about 250 mg/L, from about 20mg/L to about 200 mg/L, from 20 mg/L to about 175 mg/L, from about 20mg/L to about 150 mg/L, from about 20 mg/L to about 125 mg/L, from about20 mg/L to about 100 mg/L, from about 30 mg/L to about 300 mg/L, fromabout 30 mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L,from about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150mg/L, from about 30 mg/L to about 125 mg/L, from about 30 mg/L to about100 mg/L, from about 50 mg/L to about 300 mg/L, from about 50 mg/L toabout 250 mg/L, from about 50 mg/L to about 200 mg/L, from 50 mg/L toabout 175 mg/L, from about 50 mg/L to about 150 mg/L, from about 50 mg/Lto about 125 mg/L, or from about 50 mg/L to about 100 mg/L.

In one embodiment, the antibodies of the invention exhibit increasedstability and/or enhanced production efficiency. In one embodiment, theantibodies of the invention exhibit a production efficiency at least 2times, at least 3 times, at least 4 times, at least 5 time, at least 6times, at least 7 times, at least 8 times, at least 10 times or thatexhibited by antibody 3.19.3.

Antibody Purification and Isolation

When using recombinant techniques, the antibodies of the invention canbe produced intracellularly, in the periplasmic space, or directlysecreted into the medium. If the protein is produced intracellularly, asa first step, the particulate debris, either host cells or lysedfragments, is removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology, 10:163-167 (1992)describe a procedure for isolating antibodies which are secreted intothe periplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the antibody is secreted into themedium, supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, ion exchange chromatography, gel electrophoresis,dialysis, and/or affinity chromatography either alone or in combinationwith other purification steps. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc that is present in the antibody. Protein A can be used to purifyantibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmarket al., J. Immunol. Methods, 62:1-13 (1983)). Protein G is recommendedfor all mouse isotypes and for human γ3 (Guss et al., EMBO J.,5:15671575 (1986)). The matrix to which the affinity ligand is attachedis most often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the protein ofthe invention comprises a CH3 domain, the Bakerbond ABX resin (J.T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin, SEPHAROSE chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibodies of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, and performed at low salt concentrations (e.g.,from about 0-0.25 M salt).

Recombinant protein isolation and purification can be accomplished bymany art-accepted techniques exploiting the physical characteristics ofthe protein of interest, such as size, charge, hydrophobicity, affinity,etc. In one embodiment, the proteins of the invention are subjected toisolation/purification methods known in the art such as size exclusionchromatography, ion-exchange chromatography, and affinitychromatography. In another embodiment, the proteins of the invention arepurified through protein A affinity chromatography. In anotherembodiment, the proteins of the invention are purified through affinitychromatography exploiting one or more binding specificities within theprotein.

To ensure the stability of the antibodies of the invention, suitableassays have been developed. In one embodiment, the stability of proteinsof the invention is characterized by known techniques in the art. Inother embodiments, the stability of the proteins of the invention can beassessed by aggregation and/or fragmentation rate or profile. Todetermine the level of aggregation or fragmentation, many techniques maybe used. In one embodiment, the aggregation and/or fragmentation profilemay be assessed by the use of analytical ultracentrifugation (AUC),size-exclusion chromatography (SEC), high-performance size-exclusionchromatography (HPSEC), melting temperature (T_(m)), polyacrylamide gelelectrophoresis (PAGE), capillary gel electrophoresis (CGE), lightscattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea-induced protein unfolding techniques,intrinsic tryptophan fluorescence, differential scanning calorimetry, or1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques.In another embodiment, the stability of proteins of the invention ischaracterized by polyacrylamide gel electrophoresis (PAGE) analysis. Inanother embodiment, the stability of the proteins of the invention ischaracterized by size exclusion chromatography (SEC) profile analysis.

Another measure of stability is the relative resistance to proteasedegradation exhibited by a protein. In one embodiment, the stability ofthe proteins of the invention is characterized by a protease resistanceassay. In one embodiment, the protease utilized in the proteaseresistance assay is a serine protease, threonine protease, cysteineprotease, aspartic acid protease, metalloprotease , or a glutamic acidprotease. In one embodiment, the proteins of the invention are subjectedto a protease resistance assay in which the protease is trypsin,chymotrypsin, cathepsin B, D, L, or G, pepsin, papain, elastase, HIV-1protease, chymosin, renin, plasmepsin, plasmin, carboxypeptidase E,caspase 1-10, or calpain. In another embodiment, proteins of theinvention exhibit a low level of protease degradation. In someembodiments, the antibodies of the invention exhibit protease resistancein which at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or more of the protein remains undigested afterincubation with the protease under standard conditions for the proteaseselected.

The invention also provides methods of testing the binding of antibodiesof the invention. The binding specificities of an antibody can beassessed by many different art accepted techniques such as phage displayand other ELISA based technologies. In one embodiment, the bindingspecificities of the antibodies of the invention may be tested by anywell known technique in the art. In another embodiment, the antibodiesof the invention may be analyzed by any of the techniques presented inthe specification. In another embodiment, the binding specificities forantibodies of the invention may be tested by an ELISA based assay.

Methods of Monitoring the Stability and Aggregation of AntibodyFormulations

There are various methods available for assessing the stability ofprotein formulations based on the physical and chemical structures ofthe proteins as well as on their biological activities. For example, tostudy denaturation of proteins, methods such as charge-transferabsorption, thermal analysis, fluorescence spectroscopy, circulardichroism, NMR, rCGE (reducing capillary gel electrophoresis) and HPSEC(high performance size exclusion chromatography), are available (See,for example, Wang et al., 1988, J. of Parenteral Science & Technology42(Suppl):S4-S26).

The rCGE and HPSEC are the most common and simplest methods to assessthe formation of protein aggregates, protein degradation, and proteinfragmentation. Accordingly, the stability of the liquid formulations ofthe present invention may be assessed by these methods.

The liquid formulations of the present invention comprise an antibody ofthe invention and exhibit low to undetectable levels of aggregation asmeasured by HPSEC or rCGE, that is, no more than 5%, no more than 4%, nomore than 3%, no more than 2%, no more than 1%, or no more than 0.5%aggregate by weight protein, and low to undetectable levels offragmentation, that is, 80% or higher, 85% or higher, 90% or higher, 95%or higher, 98% or higher, or 99% or higher, or 99.5% or higher of thetotal peak area in the peak(s) representing intact antibodies. Antibodyformulations often comprise antibodies at a concentration of about 1-100mg/ml along with an appropriate excipient. These antibody formulationsmay be analyzed for aggregation levels at 1, 2, 3, 4, 5, 6, 7, 14, 21,28, 35, or 45 days or more after formulation. Also, in stabilitystudies, antibody formulations are often incubated at 2-4° C., 10-15°C., 22-27° C., 30-37° C., or 40-42° C. to assess aggregation rates. Inthe case of SDS-PAGE, the density or the radioactivity of each bandstained or labeled with radioisotope can be measured and the % densityor % radioactivity of the band representing non-degraded antibodies ofthe invention can be obtained.

In one embodiment, the antibodies of the invention exhibit a loweredaggregation rate than antibody 3.19.3. In one embodiment, the antibodiesof the invention exhibit an aggregation rate that is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 95% lower than theaggregation rate exhibited by antibody 3.19.3 assessed under similarexperimental conditions. In another embodiment, the antibodies of theinvention exhibit an aggregation rate of at least 25%, at least 20%, atleast 15%, at least 10%, at least 5%, at least 2%, at least 1%, or atleast 0.5% as measured by the experimental conditions outlined inExample 2.

The stability of the liquid formulations of the present invention can bealso assessed by any assays which measure the biological activity of theantibodies in the formulation. The biological activities of antibodiesinclude, but are not limited to, antigen-binding activity,complement-activation activity, Fc-receptor binding activity,receptor/ligand neutralizing activity, receptor agonism or antagonismand so forth. Antigen-binding activity of the antibodies can be measuredby any method known to those skilled in the art, including but notlimited to ELISA, radioimmunoassay, Western blot, and the like (Also seeHarlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988) (incorporated by reference herein in itsentirety). The purity of the liquid antibody formulations of theinvention may be measured by any method well-known to one of skill inthe art such as, e.g., HPSEC. The sterility of the liquid antibodyformulations may be assessed as follows: sterile soybean-casein digestmedium and fluid thioglycollate medium are inoculated with a test liquidantibody formulation by filtering the liquid formulation through asterile filter having a nominal porosity of 0.45 μm. When using theSterisure™ or Steritest™ method, each filter device is asepticallyfilled with approximately 100 ml of sterile soybean-casein digest mediumor fluid thioglycollate medium. When using the conventional method, thechallenged filter is aseptically transferred to 100 ml of sterilesoybean-casein digest medium or fluid thioglycollate medium. The mediaare incubated at appropriate temperatures and observed three times overa 14 day period for evidence of bacterial or fungal growth.

Methods of Using Antibodies

In addition, embodiments of the invention include methods of using theseantibodies for treating diseases. Anti-Ang-2 antibodies are useful forpreventing Ang-2 mediated Tie2 signal transduction, thereby inhibitingangiogenesis. The mechanism of action of this inhibition may includeinhibition of Ang-2/Ang-1 from binding to the receptor Tie2; inhibitionof Ang-2/Ang-1 induced Tie2 signaling; Ang-2/Ang1 mediatedphosphorylation of Tie-2; or enhanced clearance of Ang-2 thereinlowering the effective concentration of Ang-2 for binding to Tie-2. Inanother embodiment, the antibodies of the invention may act throughreducing circulating Ang-2/Ang-1 levels.

Diseases that are treatable through this inhibition mechanism include,but are not limited to, neoplastic diseases, such as, melanoma, smallcell lung cancer, non-small cell lung cancer, glioma, hepatocellular(liver) carcinoma, glioblastoma, and cancers and tumors of the thyroid,stomach, prostate, breast, ovary, bladder, lung, uterus, kidney, colon,and pancreas, salivary gland, and colorectal.

Other embodiments of the invention include diagnostic assays forspecifically determining the quantity of Ang-2 in a biological sample.The assay kit can include anti-Ang-2 antibodies along with the necessarylabels for detecting such antibodies. These diagnostic assays are usefulto screen for angiogenesis-related diseases including, but not limitedto, neoplastic diseases, such as, melanoma, small cell lung cancer,non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma,glioblastoma, and carcinoma of the thyroid, stomach, prostate, breast,ovary, bladder, lung, uterus, kidney, colon, and pancreas, salivarygland, and colorectum.

According to another aspect of the invention there is provided anantagonist of the biological activity of Angiopoietin-1 andAngiopoietin-2 wherein the antagonist binds to Angiopoietin-1 andAngiopoietin-2.

According to another aspect of the invention there is provided anantagonist of the biological activity of Angiopoietin-1 andAngiopoietin-2 wherein the antagonist is not a compound.

In one embodiment there is provided an antagonist of the biologicalactivity of Angiopoietin-1 and Angiopoietin-2 wherein the Angiopoietin-1antagonist activity and the Angiopoietin-2 antagonist activity iscomprised within one molecule. In an alternative embodiment there isprovided an antagonist wherein the Angiopoietin-1 antagonist activityand the Angiopoietin-2 antagonist activity is comprised within more thanone molecule.

In one embodiment there is provided an antagonist of the biologicalactivity of Angiopoietin-1 and Angiopoietin-2 wherein the antagonist maybind to:

-   -   I. the Tie-2 receptor;    -   II. Angiopoietin-1 and/or Angiopoietin-2;    -   III. Tie-2 receptor-Angiopoietin-1 complex; or    -   IV. Tie-2 receptor-Angiopoietin-2 complex,    -   V. or any combination of these.

In one embodiment the antagonist of the biological activity ofAngiopoietin-1 and Angiopoietin-2 may bind to Angiopoietin-1 and/orAngiopoietin-2 and/or Tie-2 and thereby prevent Angiopoietin-1 andAngiopoietin-2 mediated Tie-2 signal transduction, thereby inhibitingangiogenesis. The mechanism of action of this inhibition may include;

-   -   I. binding of the antagonist to Angiopoietin-1 and inhibiting        the binding of Angiopoietin-1 to its receptor, Tie-2, and/or    -   II. binding of the antagonist to Angiopoietin-2 and inhibit the        binding of Angiopoietin-2 to its receptor, Tie-2, and/or    -   III. enhancing the clearance of Angiopoietin-1 and/or        Angiopoietin-2 therein lowering the effective concentration of        Angiopoietin-1 and/or Angiopoietin-2 available for binding to        Tie-2,    -   IV. or any combination of these, sufficient to antagonize the        biological activity of Angiopoietin-1 and Angiopoietin-2.

Without wishing to be bound by theoretical considerations, mechanisms bywhich antagonism of the biological activity of Angiopoietin-1 and/orAngiopoietin-2 can be achieved include, but are not limited to,inhibition of binding of Angiopoietin-1 and/or Angiopoietin-2 to thereceptor Tie-2, inhibition of Angiopoietin-1 and/or Angiopoietin-2induced Tie-2 signaling, reduced Angiopoietin-1 and/or Angiopoietin-2mediated Tie-2 phosphorylation or increased clearance of Angiopoietin-1and/or Angiopoietin-2, therein reducing the effective concentration ofAngiopoietin-1 and/or Angiopoietin-2.

According to another aspect of the invention there is provided a methodof antagonizing the biological activity of Angiopoietin-1 andAngiopoietin-2 comprising administering an antagonist as describedhereinabove. The method may include selecting an animal in need oftreatment for disease-related angiogenesis, and administering to saidanimal a therapeutically effective dose of an antagonist of thebiological activity of Angiopoietin-1 and Angiopoietin-2.

According to another aspect of the invention there is provided a methodof antagonizing the biological activity of Angiopoietin-1 andAngiopoietin-2 comprising administering an antibody as describedhereinabove. The method may include selecting a subject in need oftreatment for disease-related angiogenesis, and administering to saidsubject a therapeutically effective dose of an antibody whichantagonizes the biological activity of Angiopoietin-1 andAngiopoietin-2.

According to another aspect there is provided a method of treatingdisease-related angiogenesis in a mammal comprising administering atherapeutically effective amount of an antagonist of the biologicalactivity of Angiopoietin-1 and Angiopoietin-2. The method may includeselecting a subject in need of treatment for disease-relatedangiogenesis, and administering to said subject a therapeuticallyeffective dose of an antagonist of the biological activity ofAngiopoietin-1 and Angiopoietin-2.

According to another aspect there is provided a method of treatingdisease-related angiogenesis in a subject comprising administering atherapeutically effective amount of an antibody which antagonizes thebiological activity of Angiopoietin-1 and Angiopoietin-2. The method mayinclude selecting a subject in need of treatment for disease-relatedangiogenesis, and administering to said subject a therapeuticallyeffective dose of an antibody which antagonizes the biological activityof Angiopoietin-1 and Angiopoietin-2. The antibody can be administeredalone, or can be administered in combination with additional antibodiesor chemotherapies, biological therapies/immunotherapies, radiationtherapies, hormonal therapies, or surgery.

According to another aspect there is provided a method of treatingcancer in a mammal comprising administering a therapeutically effectiveamount of an antagonist of the biological activity of Angiopoietin-1 andAngiopoietin-2. The method may include selecting an animal in need oftreatment for cancer, and administering to said animal a therapeuticallyeffective dose of an antagonist which antagonizes the biologicalactivity of Angiopoietin-1 and Angiopoietin-2. The antagonist can beadministered alone, or can be administered in combination withadditional antibodies or chemotherapies, biologicaltherapies/immunotherapies, radiation therapies, hormonal therapies, orsurgery.

According to another aspect of the invention there is provided the useof an antibody of the invention for the manufacture of a medicament forthe treatment of disease-related angiogenesis.

According to another aspect of the invention there is provided the useof an antibody which antagonizes the biological activity ofAngiopoietin-1 and Angiopoietin-2 for the manufacture of a medicamentfor the treatment of disease-related angiogenesis.

In one embodiment the present invention is particularly suitable for usein antagonizing Angiopoietin-1 or Angiopoietin-2, in patients with atumor which is dependent alone, or in part, on a Tie-2 receptor.

The invention also provides methods of using antibodies to ameliorate,treat, or prevent cancer or symptoms thereof. In one embodiment, methodsof the invention are useful in the treatment of cancers of the head,neck, eye, mouth, throat, esophagus, chest, skin, bone, lung, colon,rectum, colorectal, stomach, spleen, kidney, skeletal muscle,subcutaneous tissue, metastatic melanoma, endometrial, prostate, breast,ovaries, testicles, thyroid, blood, lymph nodes, kidney, liver,pancreas, brain, or central nervous system. Examples of cancers that canbe prevented, managed, treated or ameliorated in accordance with themethods of the invention include, but are not limited to, cancer of thehead, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon,rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, andbrain. Additional cancers include, but are not limited to, thefollowing: leukemias such as but not limited to, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias such as myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias andmyelodysplastic syndrome, chronic leukemias such as but not limited to,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone cancer andconnective tissue sarcomas such as but not limited to bone sarcoma,myeloma bone disease, multiple myeloma, cholesteatoma-induced boneosteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma,Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone,chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, andsynovial sarcoma; brain tumors such as but not limited to, glioma,astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glialtumor, acoustic neurinoma, craniopharyngioma, medulloblastoma,meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma;breast cancer including but not limited to adenocarcinoma, lobular(small cell) carcinoma, intraductal carcinoma, medullary breast cancer,mucinous breast cancer, tubular breast cancer, papillary breast cancer,Paget's disease (including juvenile Paget's disease) and inflammatorybreast cancer; adrenal cancer such as but not limited to pheochromocytomand adrenocortical carcinoma; thyroid cancer such as but not limited topapillary or follicular thyroid cancer, medullary thyroid cancer andanaplastic thyroid cancer; pancreatic cancer such as but not limited to,insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secretingtumor, and carcinoid or islet cell tumor; pituitary cancers such as butlimited to Cushing's disease, prolactin-secreting tumor, acromegaly, anddiabetes insipius; eye cancers such as but not limited to ocularmelanoma such as iris melanoma, choroidal melanoma, and cilliary bodymelanoma, and retinoblastoma; vaginal cancers such as squamous cellcarcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamouscell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,and Paget's disease; cervical cancers such as but not limited to,squamous cell carcinoma, and adenocarcinoma; uterine cancers such as butnot limited to endometrial carcinoma and uterine sarcoma; ovariancancers such as but not limited to, ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor; esophageal cancerssuch as but not limited to, squamous cancer, adenocarcinoma, adenoidcyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma,sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell(small cell) carcinoma; stomach cancers such as but not limited to,adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading,diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma, gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, adenocarcinoma,leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers suchas but not limited to squamous cell carcinoma; basal cancers; salivarygland cancers such as but not limited to adenocarcinoma, mucoepidermoidcarcinoma, and adenoidcystic carcinoma; pharynx cancers such as but notlimited to squamous cell cancer, and verrucous; skin cancers such as butnot limited to, basal cell carcinoma, squamous cell carcinoma andmelanoma, superficial spreading melanoma, nodular melanoma, lentigomalignant melanoma, acral lentiginous melanoma; kidney cancers such asbut not limited to renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesotheliorna,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas (for areview of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, InformedDecisions: The Complete Book of Cancer Diagnosis, Treatment, andRecovery, Viking Penguin, Penguin Books U.S.A., inc., United States ofAmerica). It is also contemplated that cancers caused by aberrations inapoptosis can also be treated by the methods and compositions of theinvention. Such cancers may include, but not be limited to, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis, and myelodysplastic syndromes.

The invention also provides methods of using antibodies to deplete acell population. In one embodiment, methods of the invention are usefulin the depletion of the following cell types: eosinophil, basophil,neutrophil, T cell, B cell, mast cell, monocytes, endothelial cell andtumor cell. Tumor cells may be cells derived from any of the cancerdisorders described herein.

The antibodies of the invention and compositions comprising the same areuseful for many purposes, for example, as therapeutics against a widerange of chronic and acute diseases and disorders including, but notlimited to, autoimmune and/or inflammatory disorders, which includeSjogren's syndrome, rheumatoid arthritis, lupus psoriasis,atherosclerosis, diabetic and other retinopathies, retrolentalfibroplasia, age-related macular degeneration, neovascular glaucoma,hemangiomas, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, and chronic inflammation, sepsis,rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury,septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis,arthritis (e.g., psoriatic arthritis), anaphylactic shock, organischemia, reperfusion injury, spinal cord injury and allograftrejection. Other Examples of autoimmune and/or inflammatory disordersinclude, but are not limited to, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren'ssyndrome, psoriasis, atherosclerosis, diabetic and other retinopathies,retrolental fibroplasia, age-related macular degeneration, neovascularglaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease),corneal and other tissue transplantation, and chronic inflammation,sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusioninjury, septicemia, endotoxic shock, cystic fibrosis, endocarditis,psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock,organ ischemia, reperfusion injury, spinal cord injury and allograftrejection. autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserythematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

In one embodiment disease-related angiogenesis may be bone and jointdisease: for example, but not limited to arthritis associated with orincluding osteoarthritis/osteoarthrosis, both primary and secondary to,for example, congenital hip dysplasia; cervical and lumbar spondylitis,and low back and neck pain; rheumatoid arthritis and Still's disease;seronegative spondyloarthropathies including ankylosing spondylitis,psoriatic arthritis, reactive arthritis and undifferentiatedspondarthropathy; septic arthritis and other infection-relatedarthopathies and bone disorders such as tuberculosis, including Potts'disease and Poncet's syndrome; acute and chronic crystal-inducedsynovitis including urate gout, calcium pyrophosphate depositiondisease, and calcium apatite related tendon, bursal and synovialinflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies; pain and connective tissue remodellingof musculoskeletal disorders due to injury [for example sports injury]or disease: for example arthitides (for example rheumatoid arthritis,osteoarthritis, gout or crystal arthropathy), other joint disease (suchas intervertebral disc degeneration or temporomandibular jointdegeneration), bone remodelling disease (such as osteoporosis, Paget'sdisease or osteonecrosis), polychondritits, scleroderma, reactivearthritis, polymyalgia, mixed connective tissue disorder,spondyloarthropathies or periodontal disease (such as periodontitis).

In one embodiment disease-related angiogenesis may be selected from oneof the inflammatory arthritis group of diseases including seronegativearthritis, seropositive arthritis, arthritis related to otherarthropathies, osteoarthritis or systemic lupus erythematosus (SLE). Inanother embodiment disease-related angiogenesis may be rheumatoidarthritis, seronegative spondyloarthropathy, arthritis related to otherarthropathies or SLE. In one embodiment the seronegativespondyloarthropathy is selected from ankylosing spondylitis, psoriaticarthritis, reactive arthritis or inflammatory bowel disorder-relatedarthropathies. In a specific embodiment disease-related angiogenesis isrheumatoid arthritis. In another specific embodiment disease-relatedangiogenesis is osteoarthritis.

In some embodiments, methods of the invention can be used to reduce orinhibit disease-related angiogenesis. In some embodiments, methods ofthe invention comprise a reduction or inhibition of disease-relatedangiogenesis by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75% , at least 80%, at least 85%, at least 90%, or at least 95% ofthe original disease-related angiogenesis. In other embodiments, methodsof the invention comprise a reduction or inhibition of disease-relatedangiogenesis by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75% , at least 80%, at least 85%, at least 90%, or at least 95% ofthe disease-related angiogenesis prior to treatment. In someembodiments, reduction in angiogenesis may be measured by the methodspresented herein in the Examples, or by other methods known in the art.In specific embodiments, reduction of angiogensis may be measured bystaining of biopsy samples, or by FITC-dextran accumulation in a tissue.

In some embodiments, methods of the invention can be used to reduce orinhibit angiogenesis mediated by angiogenic factors. Such factorsinclude, but are not limited to FGF, FGF2, VEGF (and various isoformsthereof), PDGF, TGF-13, endoglin, MCP-1, and ephrins. In someembodiments, methods of the invention comprise a reduction or inhibitionof angiogenesis mediated by angiogenic factors by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75% , at least 80%, at least85%, at least 90%, or at least 95% of the level of angiogenesis mediatedin the absence of treatment. In a specific embodiment, methods of theinvention reduce angiogenesis induced by at least one or more angiogenicfactor selected from the group consisting of FGF, FGF2, VEGF (andvarious isoforms thereof), PDGF, TGF-β, endoglin, MCP-1, and ephrins.

In a specific embodiment, methods of the invention can be used to reduceor inhibit FGF2-mediated angiogenesis. In some embodiments, methods ofthe invention comprise a reduction or inhibition of FGF2-mediatedangiogenesis by at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75% , at least 80%, at least 85%, at least 90%, or at least 95% ofthe level of a control FGF2-mediated angiogenesis sample.

In some embodiments, methods of the invention can be used to reduce orinhibit symptoms associated with disease-related angiogenesis. In someembodiments, methods of the invention comprise a reduction or inhibitionof symptoms associated with disease-related angiogenesis by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75% , at least 80%, atleast 85%, at least 90%, or at least 95% of the original symptomsassociated with disease-related angiogenesis. In other embodiments,methods of the invention comprise a reduction or inhibition ofdisease-related angiogenesis by at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75% , at least 80%, at least 85%, at least 90%, orat least 95% of the symptoms associated with disease-relatedangiogenesis prior to treatment. Such symptoms include swelling,inflammation, fever, pain, edema, pleural effusions, anemia, malaise,morning stiffness, lows of weight, poor circulation, numbness in thelimbs, and the like.

In other embodiments, methods of the invention can be used to maintaindisease-related angiogenesis. In some embodiments, methods of theinvention can be used to prevent disease-related angiogenesis.

The compositions and methods of the invention can be used with one ormore conventional therapies that are used to prevent, manage or treatthe above diseases.

In one embodiment, the invention provides a method of preventingdevelopment of cancer from a pre-cancerous state in an animal. Thepre-cancerous state may be dysplasia, hyperplasia or cancer in situ(cancer in place).

In another embodiment, the invention provides a method of treating,preventing, ameliorating or managing symptoms of cancer in an animal.Symptoms of cancer may vary greatly depending on the nature of thecancer and state of progression. Symptoms associated with some cancertypes may include; Bladder cancer: blood in the urine, pain or burningupon urination; frequent urination; or cloudy urine; Bone cancer: painin the bone or swelling around the affected site; fractures in bones;weakness, fatigue; weight loss; repeated infections; nausea, vomiting,constipation, problems with urination; weakness or numbness in the legs;bumps and bruises that persist; Brain cancer: dizziness; drowsiness;abnormal eye movements or changes in vision; weakness, loss of feelingin arms or legs or difficulties in walking; fits or convulsions; changesin personality, memory or speech; headaches that tend to be worse in themorning and ease during the day, that may be accompanied by nausea orvomiting; Breast cancer: a lump or thickening of the breast; dischargefrom the nipple; change in the skin of the breast; a feeling of heat; orenlarged lymph nodes under the arm; Colorectal cancer: rectal bleeding(red blood in stools or black stools); abdominal cramps; constipationalternating with diarrhea; weight loss; loss of appetite; weakness;pallid complexion; Kidney cancer: blood in urine; dull ache or pain inthe back or side; lump in kidney area, sometimes accompanied by highblood pressure or abnormality in red blood cell count; Leukemia:weakness, paleness; fever and flu-like symptoms; bruising and prolongedbleeding; enlarged lymph nodes, spleen, liver; pain in bones and joints;frequent infections; weight loss; night sweats; Lung cancer: wheezing,persistent cough for months; blood-streaked sputum; persistent ache inchest; congestion in lungs; enlarged lymph nodes in the neck; Melanoma:change in mole or other bump on the skin, including bleeding or changein size, shape, color, or texture; Non-Hodgkin's lymphoma: painlessswelling in the lymph nodes in the neck, underarm, or groin; persistentfever; feeling of fatigue; unexplained weight loss; itchy skin andrashes; small lumps in skin; bone pain; swelling in the abdomen; liveror spleen enlargement; Oral cancer: a lump in the mouth, ulceration ofthe lip, tongue or inside of the mouth that does not heal within acouple of weeks; dentures that no longer fit well; oral pain, bleeding,foul breath, loose teeth, and changes in speech; Ovarian cancer:abdominal swelling; in rare cases, abnormal vaginal bleeding; digestivediscomfort; Pancreatic cancer: upper abdominal pain and unexplainedweight loss; pain near the center of the back; intolerance of fattyfoods; yellowing of the skin; abdominal masses; enlargement of liver andspleen; Prostate cancer: urination difficulties due to blockage of theurethra; bladder retains urine, creating frequent feelings of urgency tourinate, especially at night; bladder not emptying completely; burningor painful urination; bloody urine; tenderness over the bladder; anddull ache in the pelvis or back; Stomach cancer: indigestion orheartburn; discomfort or pain in the abdomen; nausea and vomiting;diarrhea or constipation; bloating after meals; loss of appetite;weakness and fatigue; bleeding—vomiting blood or blood in the stool;Uterine cancer: abnormal vaginal bleeding, a watery bloody discharge inpostmenopausal women; a painful urination; pain during intercourse; painin pelvic area.

In another embodiment, the invention provides a method of promotingtumor regression of a cancer. In one embodiment, the method involvesregression of the tumor by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75% , at least 80%, at least 85%, at least 90%, atleast 95% of the original tumor size. In a specific embodiment, themethod comprises the elimination of the tumor by inhibition ofangiogenesis.

Cell proliferation rates may be assayed by many means known in the artsuch as thymidine incorporation, DNA content, or cell counts. In anotherembodiment, the invention provides a method of inhibiting tumor cellproliferation. In one embodiment the method comprises a reduction oftumor cell proliferation. In another embodiment, the method comprises areduction of tumor cell proliferation by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75% , at least 80%, at least 85%, atleast 90%, at least 95% of the original tumor cell proliferation rate.

In another embodiment the invention provides a method of depletingmalignant tumor cells. In some embodiments, the method comprisesdepleting tumor cells resident in the primary tumor. In otherembodiments, the method comprises depleting tumor cells in circulation.In other embodiments, the method comprises depleting tumor cells in asecondary site.

In another embodiment, the invention provides a method of inhibitingangiogenesis of a cancer tumor. In some embodiments, the methodcomprises inhibition of angiogenesis of a primary tumor site. In otherembodiments, the method comprises inhibition of angiogenesis of asecondary tumor site.

The present invention provides methods of preventing, treating, managingor ameliorating an inflammatory disorder or an autoimmune disorder orone or more symptoms thereof in a subject, said methods comprisingadministering to said subject an antibody of the invention. In someembodiments, the present invention provides methods of preventing,treating, managing or ameliorating an inflammatory disorder or anautoimmune disorder associated with inflammation or one or more symptomsthereof in a subject, said methods comprising administering to saidsubject an antibody of the invention and one or more TNFα antagonists.In further embodiments, at least one of the TNF-α antagonists is asoluble TNF-α receptor such as etanercept (ENBREL™; Immunex) or afragment, derivative or analog thereof, or an antibody thatimmunospecifically binds to TNF-α such as infliximab (REMICADE™;Centocor) or adalimumab (HUMIRA™; Abbott) a derivative, analog orantigen-binding fragment thereof. In specific embodiments the methods ofthe invention are performed prophylactically or therapeutically.

In some embodiments, the present invention provides methods ofpreventing, treating, managing or ameliorating at least one symptomassociated with an inflammatory or autoimmune disorder. Such symptomsmay include anemia, swelling, inflammation, edema, rash, swelling in thejoints, bone synovial hyperplasia, synovitis, synovial fibrosis,periostitis, or bone mineral density (loss). In some embodiments, thepresent invention provides methods of treating or managing at least onesymptom in an individual associated with an inflammatory or autoimmunedisorder wherein said method results in a reduction of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or more over the severity of atleast one symptom in an individual in the absence of treatment.

Combinations

The anti-angiogenic treatment defined herein may be applied as a soletherapy or may involve, in addition to the compounds of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti tumoragents:

(i) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), estrogen receptordown-regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5′ reductase such as finasteride;

(ii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iii) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-ErbB2 antibody trastuzumab [Herceptin™] and theanti-ErbB1 antibody cetuximab [C225]), farnesyl transferase inhibitors,tyrosine kinase inhibitors and serine/threonine kinase inhibitors, forexample inhibitors of the epidermal growth factor family (for exampleEGFR family tyrosine kinase inhibitors such as N (3-chloro4-fluorophenyl) 7-methoxy 6 (3-morpholinopropoxy)quinazolin 4-amine(gefitinib, AZD1839), N (3-ethynylphenyl) 6,7bis(2-methoxyethoxy)quinazolin 4-amine (erlotinib, OSI 774) and6-acrylamido N (3-chloro 4-fluorophenyl) 7(3-morpholinopropoxy)quinazolin 4-amine (CI 1033)), for exampleinhibitors of the platelet derived growth factor family and for exampleinhibitors of the hepatocyte growth factor family;

(iv) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti vascularendothelial cell growth factor antibody bevacizumab (Avastin®),anti-vascular endothelial growth factor receptor antibodies suchanti-KDR antibodies and anti-fltl antibodies, compounds such as thosedisclosed in International Patent Applications WO 97/22596, WO 97/30035,WO 97/3285, WO 98/13354, WO00/47212 and WO01/32651) and compounds thatwork by other mechanisms (for example linomide, inhibitors of integrinαvβ3 function and angiostatin);

(v) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vi) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;

(vii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene directed enzyme pro drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi drug resistance gene therapy; and

(viii) immunotherapy approaches, including for example ex vivo and invivo approaches to increase the immunogenicity of patient tumor cells,such as transfection with cytokines such as interleukin-2, interleukin-4or granulocyte macrophage colony stimulating factor, approaches todecrease T cell anergy, approaches using transfected immune cells suchas cytokine transfected dendritic cells, approaches using cytokinetransfected tumor cell lines and approaches using anti idiotypicantibodies.

In one embodiment of the invention the anti-angiogenic treatments of theinvention are combined with agents which inhibit the effects of vascularendothelial growth factor (VEGF), (for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin®),anti-vascular endothelial growth factor receptor antibodies suchanti-KDR antibodies and anti-flt1 antibodies, compounds such as thosedisclosed in International Patent Applications WO 97/22596, WO 97/30035,WO 97/3285, WO 98/13354, WO00/47212 and WO01/32651) and compounds thatwork by other mechanisms (for example linomide, inhibitors of integrinavb3 function and angiostatin); In another embodiment of the inventionthe anti-angiogenic treatments of the invention are combined agentswhich inhibit the tyrosine kinase activity of the vascular endothelialgrowth factor receptor, KDR (for example AZD2171 or AZD6474). Additionaldetails on AZD2171 may be found in Wedge et al (2005) Cancer Research.65(10):4389-400. Additional details on AZD6474 may be found in Ryan &Wedge (2005) British Journal of Cancer. 92 Suppl 1:S6-13. Bothpublications are herein incorporated by reference in their entireties.In another embodiment of the invention the fully human antibodies3.19.3, 3.3.2 or 5.88.3 are combined alone or in combination withAvastin®, AZD2171 or AZD6474.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the compounds of thisinvention, or pharmaceutically acceptable salts thereof, within thedosage range described hereinbefore and the other pharmaceuticallyactive agent within its approved dosage range.

Combinations of Ang2 Antagonists and Chemotherapy Agents

We have found that certain combinations of an antagonist of thebiological activity of Angiopoietin-2 (including, but not limited tomonoclonal antibody 3.19.3) and a chemotherapeutic agent producessignificantly better effects on tumors as compared to use of theantagonist of the biological activity of Angiopoietin-2 or achemotherapeutic agent used alone.

Accordingly, embodiments of the present invention provide methods ofproduction of an anti-cancer effect in a patient, which comprisesadministering to said patient a therapeutically effective amount of anantagonist of the biological activity Angiopoietin-2, and/or Tie-2,before, after or simultaneously with an effective amount of achemotherapeutic agent. In some embodiments, the method comprisesselecting a patient in need of an anti-cancer effect, and administeringto the patient a therapeutically effective dose of a combination of anantagonist of the biological activity of Angiopoietin-2, and/or Tie-2,and a chemotherapeutic agent.

In other embodiments, methods of the invention comprise the productionof an antiangiogenic and/or vascular permeability reducing effect in apatient which comprises administering to said patient an effectiveamount of an antagonist of the biological activity of Angiopoietin-2,and/or Tie-2, before, after or simultaneously with an effective amountof a chemotherapeutic agent. In some embodiments, the method comprisesselecting a patient in need an antiangiogenic and/or vascularpermeability reducing effect, and administering to the patient atherapeutically effective dose of a combination of an antagonist of thebiological activity of Angiopoietin-2, and/or Tie-2, and achemotherapeutic agent.

In other embodiments, the invention provides a method for the treatmentof disease-related angiogenesis in a patient which comprisesadministering to said patient an effective amount of an antagonist ofthe biological activity of Angiopoietin-2, and/or Tie-2, before, afteror simultaneously with an effective amount of a chemotherapeutic agent.In some embodiments, the method comprises selecting a patient in need oftreatment of disease-related angiogenesis, and administering to thepatient a therapeutically effective dose of a combination of anantagonist of the biological activity of Angiopoietin-2, and/or Tie-2,and a chemotherapeutic agent.

In other embodiments, the invention provides a method of antagonizingthe biological activity of Angiopoietin-2, and/or Tie-2 in a patient,which comprises administering to said patient in need thereof aneffective amount of an antagonist of the biological activityAngiopoietin-2, and/or Tie-2, before, after or simultaneously with aneffective amount of a chemotherapeutic agent.

According to a further aspect of the present invention there is provideda method of treatment comprising the administration of an effectiveamount of an antagonist of the biological activity of Angiopoietin-2,and/or Tie-2, or a pharmaceutically acceptable salt thereof, optionallytogether with a pharmaceutically acceptable excipient or carrier, andthe simultaneous, sequential or separate administration of an effectiveamount of a chemotherapeutic agent or a pharmaceutically acceptable saltthereof, wherein the latter may optionally be administered together witha pharmaceutically acceptable excipient or carrier, to a patient in needof such therapeutic treatment.

In one embodiment the antagonist of the biological activity ofAngiopoietin-2 is an antibody. In further embodiments, the antagonist ofAngiopoietin-2 is a monoclonal antibody. In yet further embodiments, theantagonist of Angiopoietin-2 is a fully human monoclonal antibody. Insome embodiments the fully human monoclonal antibody is selected fromany one of: 3.31.2, or 5.16.3, or 5.86.1, or 5.88.3, or 3.3.2, or5.103.1, or 5.101.1, or 3.19.3, or 5.28.1, or 5.78.3, MEDI1/5, MEDI2/5,MEDI3/5, MEDI6/5, or MEDI4/5. In further embodiments, the fully humanmonoclonal antibody binds to the same epitope as any one of fully humanmonoclonal antibody: 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,5.101.1, 3.19.3, 5.28.1, 5.78.3 which are disclosed in InternationalPublication Number WO2006/068953 or AMG 386 (Amgen, InternationalPublication Number WO200330833).

In another embodiment the antagonist of the biological activity ofAngiopoietin-2 is a peptibody such as the peptibody (AMG386) asdisclosed in International Publication Number WO2003057134.

In another embodiment the antagonist of the biological activity of Tie-2is an antibody. In further embodiments, the Tie-2 antibody is amonoclonal, humanized, or fully human antibody.

In one embodiment a chemotherapeutic agent comprises alkylating agents(for example cisplatin, carboplatin, oxaliplatin, cyclophosphamide,nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas);antimetabolites (for example antifolates such as fluoropyrimidines like5-fluorouracil and tegafur, raltitrexed, gemcitabine, capecitabine,methotrexate, pemetrexed (Alimta), cytosine arabinoside and hydroxyurea,or, for example, one of the antimetabolites disclosed in European PatentApplication No. 562734 such as(2S)-2-{o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)butyricacid); pharmaceutical combinations which comprise an alkylating agentand an antimetabolite (for example Folfox (a combination of fluorouracil(5FU), leucovorin and oxaliplatin)); antitumor antibiotics (for exampleanthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin,epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin);antimitotic agents (for example vinca alkaloids like vincristine,vinblastine, vindesine and vinorelbine and taxoids like taxol andtaxotere); topoisomerase inhibitors (for example epipodophyllotoxinslike etoposide and teniposide, irinotecan, amsacrine, topotecan andcamptothecin); or proteasome inhibitors (for example bortezomib). In oneembodiment there is provided a combination of the invention additionallycomprising Folfox.

In another embodiment a chemotherapeutic agent comprises docetaxel, andother antimitotic agents (for example vinca alkaloids like vincristine,vinblastine, vindesine and vinorelbine and taxoids like taxol andtaxotere); 5-fluorouracil, gemcitabine and other antimetabolites (forexample antifolates such as fluoropyrimidines, tegafur, raltitrexed,capecitabine, methotrexate, pemetrexed (Alimta), cytosine arabinosideand hydroxyurea); irinotecan and other topoisomerase inhibitors (forexample etoposide topotecan, camptothecin teniposide, and amsacrine);oxaliplatin and other alkylating or DNA binding agents (for examplecis-platin, and carboplatin). In one embodiment there is provided acombination of the invention additionally comprising Folfox.

In another embodiment a chemotherapeutic agent comprises Eg5 inhibitors,for example AZD4877.

Combinations of Ang2 Antagonists and CSF1/CSFR1 Antagonists

The present invention also provides pharmaceutical combinationscomprising an antagonist of the biological activity of Angiopoietin-2,and/or Tie-2, and an antagonist of the biological activity of CSF1R,and/or CSF1, and uses of such combinations.

According to one aspect of the invention there is provided apharmaceutical combination comprising an antagonist of the biologicalactivity of Angiopoietin-2, and/or Tie-2 and an antagonist of thebiological activity of CSF1R, and/or CSF1.

In one embodiment the antagonist of the biological activity ofAngiopoietin-2 is an antibody. In further embodiments, the antagonist ofAngiopoietin-2 is a monoclonal antibody. In yet further embodiments, theantagonist of Angiopoietin-2 is a fully human monoclonal antibody. Insome embodiments the fully human monoclonal antibody is selected fromany one of; 3.31.2, or 5.16.3, or 5.86.1, or 5.88.3, or 3.3.2, or5.103.1, or 5.101.1, or 3.19.3, or 5.28.1, or 5.78.3, MEDI1/5, MEDI2/5,MEDI3/5, MEDI6/5, or MEDI4/5. In further embodiments, the fully humanmonoclonal antibody binds to the same epitope as any one of fully humanmonoclonal antibody; 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,5.101.1, 3.19.3, 5.28.1, 5.78.3 which are disclosed in InternationalPublication Number WO2006/068953 or AMG 386 (Amgen, InternationalPublication Number WO200330833).

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofTie-2 is an antibody. In one embodiment the antagonist is a monoclonalantibody. In one embodiment the antagonist is a fully human monoclonalantibody.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofCSF1R is an antibody. In one embodiment the antagonist is a monoclonalantibody. In one embodiment the antagonist is a fully human monoclonalantibody.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofCSF1 is an antibody. In one embodiment the antagonist is a monoclonalantibody. In one embodiment the antagonist is monoclonal antibodyPD-360324 (Pfizer). In one embodiment the antagonist is a fully humanmonoclonal antibody.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofCSF1R is a compound, or a pharmaceutically acceptable salt thereof. Inone embodiment the antagonist is a tyrosine kinase inhibitor, or apharmaceutically acceptable salt thereof. In one embodiment the tyrosinekinase inhibitor, or a pharmaceutically acceptable salt thereof, isselected from the compounds disclosed in International PatentApplication No.s WO2004/004985, WO02007/119046, WO2008/056148 orWO2008/090353, ABT-869 (Abbott), Sutent (Pfizer), KI-20227 (KirinBrewery), CYC-10268 (Cytopia), YM-359445 (Astellas Pharma), PLX-647(Phenomix Corp./Plexxikon), JNJ-27301937 (Johnson & Johnson), GW-2580(GlaxoSmithKline) or any of the compounds disclosed in U.S. ProvisionalApplication Numbers US05/0131022, US05/0113566, International PatentApplication Numbers WO2004/096795 WO2005/009967, WO2006/047277,WO2006/047504 or WO2003/093238.

In one embodiment the tyrosine kinase inhibitor, or a pharmaceuticallyacceptable salt thereof, is selected from the compounds disclosed inInternational Patent Application No. WO2004/004985, WO2007/119046, WO2008/090353, WO 2008/056148, WO 2007/119046, WO 2007/071955 each ofwhich are incorporated by reference in their entireties.

In another embodiment the antagonist of the biological activity of CSF1Ris selected from any one of:

-   -   2-chloro-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-benzamide;    -   2-methyl-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-pyridin-3-ylbenzamide;    -   2-chloro-5-[(3-cyclopropylbenzoyl)amino]-N-pyridin-3-ylbenzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-pyridin-3-ylbenzamide;    -   5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-[(3-chlorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-N-1,3-thiazol-5-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;    -   2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;    -   5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-1,3-thiazol-5-ylbenzamide;    -   2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methylbenzoyl)amino]-N-methyl-1,3-thiazole-2-carboxamide;    -   5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-chlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[3,4-dichlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-cyclopropylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3,5-dimethylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-methyl-5-[(3-methylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2,6-dichloro-N-(4-methyl-3-{[(2-methyl-1,3-thiazol-5-yl)amino]carbonyl}phenyl)isonicotinamide;    -   2-methyl-5-{[(3-methylcyclohexyl)carbonyl]amino}-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-(pentanoylamino)benzamide;    -   2-methyl-5-[(4-methylhexanoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2,3-dichlorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(3-chloro-2-fluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-5-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-7-(2-methoxyethoxy)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-isopropylpiperidin-4-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;        and    -   4-[(3-chloro-2-fluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;    -   7-Ethoxy-4-[(2-fluoro-4-methyl-phenyl)amino]-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   4-(2-Fluoro-4-methylphenylamino)-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   4-[(2,4-Difluorophenyl)amino]-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   6-[(3R,5        S)-3,5-Dimethylpiperazin-1-yl]-4-[(2-fluoro-4-methylphenyl)amino]-7-methoxycinnoline-3-carboxamide;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-7-methoxycinnoline-3-carboxamide;    -   7-Ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]cinnoline-3-carboxamide;    -   4-[(3-Chloro-2-fluorophenyl)amino]-6-[(3R,5        S)-3,5-dimethylpiperazin-1-yl]-7-methoxycinnoline-3-carboxamide;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxycinnoline-3-carboxamide        hydrochloride;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-[1-(2-hydroxyethyl)piperidin-4-yl]-7-methoxycinnoline-3-carboxamide;        and    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-{4-[(2R)-2-hydroxypropanoyl]piperazin-1-yl}-7-methoxycinnoline-3-carboxamide        or a pharmaceutically acceptable salt thereof.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofCSF1 is a compound, or a pharmaceutically acceptable salt thereof.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofAngiopoietin-2 is a compound, or a pharmaceutically acceptable saltthereof.

In another embodiment there is provided a pharmaceutical combination asdescribed above, wherein the antagonist of the biological activity ofTie-2 is a compound, or a pharmaceutically acceptable salt thereof. Inone embodiment the antagonist is a tyrosine kinase inhibitor, or apharmaceutically acceptable salt thereof. In one embodiment the tyrosinekinase inhibitor, or a pharmaceutically acceptable salt thereof, isselected from any of the compounds disclosed in International PatentApplication Numbers WO2004/013141, WO2004/058776, WO2005/060970 orWO2005/060969, or is GW697465X (GSK), CP-547632 (Pfizer), CE-245677(Pfizer) or CGI1631 (Cellular Genomics).

In another aspect of the present invention there is provided apharmaceutical combination of the present invention for use as amedicament comprising an antagonist of the biological activity ofAngiopoietin-2, and/or Tie-2, and an antagonist of the biologicalactivity of CSF1R, and/or CSF1, for use simultaneously, serially orseparately.

In another aspect of the present invention there is provided a method ofantagonizing Angiopoietin-2 and/or Tie-2, and antagonizing CSF1R, and/orCSF1, in a patient, which comprises administering to the patient atherapeutically effective amount of a pharmaceutical combination oranother composition of the present invention. In one embodiment themethod additionally comprises selecting a patient in need of inhibitionof Angiopoietin-2 and/or Tie-2, and inhibition of CSF1R, and/or CSF1,and administering to the patient a therapeutically effective dose of apharmaceutical combination or another pharmaceutical composition asdescribed herein.

In one embodiment the present invention is particularly suitable for usein antagonizing the biological activity of Angiopoietin-2, and/or Tie-2,and the biological activity of CSF1R, and/or CSF1, in patients with atumor which is dependent alone, or in part, on Angiopoietin-2, and/orTie-2, and CSF1R, and/or CSF1.

In one embodiment the method or use of the invention may be administeredwith one or more of the following agents, by way of the simultaneous,sequential or separate administration with of the antagonist of thebiological activity of Angiopoietin-2, and/or Tie-2, an antagonist ofcytokine function, (e.g. an agent which act on cytokine signallingpathways such as a modulator of the SOCS system), such as an alpha-,beta-, and/or gamma-interferon; modulators of insulin-like growth factortype I (IGF-1), its receptors and associated binding proteins;interleukins (IL) e.g. one or more of IL-1 to 33, and/or an interleukinantagonist or inhibitor such as anakinra; inhibitors of receptors ofinterleukin family members or inhibitors of specific subunits of suchreceptors; a tumor necrosis factor alpha (TNF-α) inhibitor such as ananti-TNF monoclonal antibody (for example infliximab; adalimumab, and/orCDP-870), and/or a TNF receptor antagonist e.g. an immunoglobulinmolecule (such as etanercept) and/or a low-molecular-weight agent suchas pentoxyfylline; a modulator of B cells, e.g. a monoclonal antibodytargeting B-lymphocytes (such as CD20 (rituximab) or MRA-aIL16R) orT-lymphocytes (e.g. CTLA4-Ig, HuMax Il-15 or Abatacept); a modulatorthat inhibits osteoclast activity, for example an antibody to RANKL; amodulator of chemokine or chemokine receptor function such as anantagonist of CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7,CCR8, CCR9, CCR10 and CCR11 (for the C—C family); CXCR1, CXCR2, CXCR3,CXCR4 and CXCR5 and CXCR6 (for the C—X—C family) and CX₃CR1 for theC—X₃—C family; antiangiogenic agents such as those which inhibit theeffects of vascular endothelial growth factor, [for example theanti-vascular endothelial cell growth factor antibody bevacizumab(Avastin®) and VEGF receptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), compounds such as thosedisclosed in International Patent Applications WO97/22596, WO 97/30035,WO 97/32856, WO 98/13354, WO00/47212 and WO01/32651 and compounds thatwork by other mechanisms (for example linomide, inhibitors of integrinαvβ3 function and angiostatin)] or colony stimulating factor 1 (CSF1) orCSF1 receptor; an inhibitor of matrix metalloproteases (MMPs), i.e., oneor more of the stromelysins, the collagenases, and the gelatinases, aswell as aggrecanase; especially collagenase-1 (MMP1), collagenase-2(MMP8), collagenase-3 (MMP13), stromelysin-1 (MMP3), stromelysin-2(MMP10), and/or stromelysin-3 (MMP11) and/or MMP9 and/or MMP12, e.g. anagent such as doxycycline; a leukotriene biosynthesis inhibitor,5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating protein(FLAP) antagonist such as; zileuton; ABT-761; fenleuton; tepoxalin;Abbott-79175; Abbott-85761;N-(5-substituted)-thiophene-2-alkylsulfonamides;2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such asZeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted2-cyanonaphthalene compound such as L-739,010; a 2-cyanoquinolinecompound such as L-746,530; indole and/or a quinoline compound such asMK-591, MK-886, and/or BAY×1005; a receptor antagonist for leukotrienes(LT) B4, LTC4, LTD4, and LTE4, selected from the group consisting of thephenothiazin-3-1s such as L-651,392; amidino compounds such asCGS-25019c; benzoxalamines such as ontazolast; benzenecarboximidamidessuch as BIIL 284/260; and compounds such as zafirlukast, ablukast,montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro-245913,iralukast (CGP 45715A), and BAY×7195; a phosphodiesterase (PDE)inhibitor such as a methylxanthanine, e.g. theophylline and/oraminophylline; and/or a selective PDE isoenzyme inhibitor e.g. a PDE4inhibitor and/or inhibitor of the isoform PDE4D, and/or an inhibitor ofPDE5; a histamine type 1 receptor antagonist such as cetirizine,loratadine, desloratadine, fexofenadine, acrivastine, terfenadine,astemizole, azelastine, levocabastine, chlorpheniramine, promethazine,cyclizine, and/or mizolastine (generally applied orally, topically orparenterally);a proton pump inhibitor (such as omeprazole) orgastroprotective histamine type 2 receptor antagonist; an antagonist ofthe histamine type 4 receptor; an alpha-1/alpha-2 adrenoceptor agonistvasoconstrictor sympathomimetic agent, such as propylhexedrine,phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine,naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozolinehydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride,and ethylnorepinephrine hydrochloride; an anticholinergic agent, e.g. amuscarinic receptor (M1, M2, and M3) antagonist such as atropine,hyoscine, glycopyrrrolate, ipratropium bromide, tiotropium bromide,oxitropium bromide, pirenzepine, and telenzepine; a beta-adrenoceptoragonist (including beta receptor subtypes 1-4) such as isoprenaline,salbutamol, formoterol, salmeterol, terbutaline, orciprenaline,bitolterol mesylate, and/or pirbuterol , e.g. a chiral enantiomerthereof; a chromone, e.g. sodium cromoglycate and/or nedocromil sodium;a glucocorticoid, such as flunisolide, triamcinolone acetonide,beclomethasone dipropionate, budesonide, fluticasone propionate,ciclesonide, and/or mometasone furoate; an agent that modulates nuclearhormone receptors such as a PPAR; an immunoglobulin (Ig) or Igpreparation or an antagonist or antibody modulating Ig function such asanti-IgE (e.g. omalizumab); other systemic or topically-appliedanti-inflammatory agent, e.g. thalidomide or a derivative thereof, aretinoid, dithranol, and/or calcipotriol; pharmaceutical combinations ofaminosalicylates and sulfapyridine such as sulfasalazine, mesalazine,balsalazide, and olsalazine; and immunomodulatory agents such as thethiopurines, and corticosteroids such as budesonide; an antibacterialagent e.g. a penicillin derivative, a tetracycline, a macrolide, abeta-lactam, a fluoroquinolone, metronidazole, and/or an inhaledaminoglycoside; and/or an antiviral agent e.g. acyclovir, famciclovir,valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine;ribavirin; zanamavir and/or oseltamavir; a protease inhibitor such asindinavir, nelfinavir, ritonavir, and/or saquinavir; a nucleosidereverse transcriptase inhibitor such as didanosine, lamivudine,stavudine, zalcitabine, zidovudine; a non-nucleoside reversetranscriptase inhibitor such as nevirapine, efavirenz; a cardiovascularagent such as a calcium channel blocker, beta-adrenoceptor blocker,angiotensin-converting enzyme (ACE) inhibitor, angiotensin-2 receptorantagonist; lipid lowering agent such as a statin, and/or fibrate; amodulator of blood cell morphology such as pentoxyfylline; athrombolytic, and/or an anticoagulant e.g. a platelet aggregationinhibitor; a CNS agent such as an antidepressant (such as sertraline),anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole,pramipexole, MAOB inhibitor such as selegine and rasagiline, comPinhibitor such as tasmar, A-2 inhibitor, dopamine reuptake inhibitor,NMDA antagonist, nicotine agonist, dopamine agonist and/or inhibitor ofneuronal nitric oxide synthase), and an anti-Alzheimer's drug such asdonepezil, rivastigmine, tacrine, COX-2 inhibitor, propentofylline ormetrifonate; an agent for the treatment of acute and chronic pain, e.g.a centrally or peripherally-acting analgesic such as an opioid analogueor derivative, carbamazepine, phenytoin, sodium valproate, amitryptilineor other antidepressant agent, paracetamol, or non-steroidalanti-inflammatory agent; a parenterally or topically-applied (includinginhaled) local anaesthetic agent such as lignocaine or an analoguethereof; an anti-osteoporosis agent e.g. a hormonal agent such asraloxifene, or a biphosphonate such as alendronate; (i) a tryptaseinhibitor; (ii) a platelet activating factor (PAF) antagonist; (iii) aninterleukin converting enzyme (ICE) inhibitor; (iv) an IMPDH inhibitor;(v) an adhesion molecule inhibitors including VLA-4 antagonist; (vi) acathepsin; (vii) a kinase inhibitor e.g. an inhibitor of tyrosinekinases (such as Btk, Itk, Jak3 MAP examples of inhibitors might includeGefitinib, Imatinib mesylate), a serine/threonine kinase (e.g. aninhibitor of MAP kinase such as p38, JNK, protein kinases A, B and C andIKK), or a kinase involved in cell cycle regulation (e.g. a cylindependent kinase); (viii) a glucose-6 phosphate dehydrogenase inhibitor;(ix) a kinin-B.sub1.- and/or B.sub2.-receptor antagonist; (x) ananti-gout agent, e.g., colchicine; (xi) a xanthine oxidase inhibitor,e.g., allopurinol; (xii) a uricosuric agent, e.g., probenecid,sulfinpyrazone, and/or benzbromarone; (xiii) a growth hormonesecretagogue; (xiv) transforming growth factor (TGFβ3); (xv)platelet-derived growth factor (PDGF); (xvi) fibroblast growth factor,e.g., basic fibroblast growth factor (bFGF); (xvii) granulocytemacrophage colony stimulating factor (GM-CSF); (xviii) capsaicin cream;(xix) a tachykinin NK.sub1. and/or NK.sub3. receptor antagonist suchNKP-608C, SB-233412 (talnetant), and/or D-4418; (xx) an elastaseinhibitor e.g. UT-77 and/or ZD-0892; (xxi) a TNF-alpha converting enzymeinhibitor (TACE); (xxii) induced nitric oxide synthase (iNOS) inhibitoror (xxiii) a chemoattractant receptor-homologous molecule expressed onTH2 cells, (such as a CRTH2 antagonist) (xxiv) an inhibitor of a P38(xxv) agent modulating the function of Toll-like receptors (TLR) and(xxvi) an agent modulating the activity of purinergic receptors such asP2X7; (xxvii) an inhibitor of transcription factor activation such asNFkB, API, and/or STATS; Non-steroidal anti-inflammatory agents(hereinafter NSAIDs) including non-selective cyclo-oxygenase(COX)-1/COX-2 inhibitors whether applied topically or systemically (suchas piroxicam, diclofenac, propionic acids such as naproxen,flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such asmefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones suchas phenylbutazone, salicylates such as aspirin); selective COX-2inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib,lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenase inhibitingnitric oxide donors (CINODs); glucocorticosteroids (whether administeredby topical, oral, intramuscular, intravenous, or intra-articularroutes); methotrexate, leflunomide; hydroxychloroquine, d-penicillamine,auranofin or other parenteral or oral gold preparations ; analgesics;diacerein; intra-articular therapies such as hyaluronic acidderivatives; and nutritional supplements such as glucosamine.

In some embodiments, the method or use of the invention may compriseadministration of an agent that antagonizes TNF-α. Any TNF-α antagonistwell-known to one of skill in the art can be used in the compositionsand methods of the invention. Non-limiting examples of TNF-α antagonistsinclude proteins, polypeptides, peptides, fusion proteins, antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab fragments, F(ab)₂ fragments, and antigen-binding fragments thereof)such as antibodies that immunospecifically bind to TNF-α, nucleic acidmolecules (e.g., antisense molecules or triple helices), organicmolecules, inorganic molecules, and small molecules that blocks,reduces, inhibits or neutralizes the function, activity and/orexpression of TNF-α. In various embodiments, a TNF-α antagonist reducesthe function, activity and/or expression of TNF-α by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% relative to a control such asphosphate buffered saline (PBS).

Examples of antibodies that immunospecifically bind to TNF-α include,but are not limited to, infliximab (REMICADE™; Centocor), adalimumab(HUMIRA™; Abbott Laboratories), D2E7 (Abbott Laboratories/KnollPharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known asHUMICADE™ and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), andTN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91:2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA89:7375-7379). The present invention also encompasses the use ofantibodies that immunospecifically bind to TNF-α disclosed in thefollowing U.S. patents in the compositions and methods of the invention:U.S. Pat. Nos. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380;5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272;5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452;5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and6,171,787; each of which are herein incorporated by reference in theirentirety. Examples of soluble TNF-α receptors include, but are notlimited to, sTNF-R1 (Amgen), etanercept (ENBREL™; Immunex) and its rathomolog RENBREL™, soluble inhibitors of TNF-α derived from TNFrI, TNFrII(Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335), and TNF-αInh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192).

In one embodiment, a TNF-α antagonist used in the compositions andmethods of the invention is a soluble TNF-α receptor. In a specificembodiment, a TNF-α antagonist used in the compositions and methods ofthe invention is etanercept (ENBREL™; Immunex) or a fragment, derivativeor analog thereof. In another embodiment, a TNF-α antagonist used in thecompositions and methods of the invention is an antibody thatimmunospecifically binds to TNF-α. In a specific embodiment, a TNF-αantagonist used in the compositions and methods of the invention isinfliximab (REMICADE™; Centocor) a derivative, analog or antigen-bindingfragment thereof. In another specific embodiment, a TNF-α antagonistused in the compositions and methods of the invention is adalimumab(HUMIRA™; Abbott Laboratories) a derivative, analog or antigen-bindingfragment thereof.

Other TNF-α antagonists encompassed by the invention include, but arenot limited to, IL-10, which is known to block TNF-α production viainterferon γ-activated macrophages (Oswald et al. 1992, Proc. Natl.Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc.Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-1(Serono/Yeda), the vaccine CytoTAb (Protherics), antisensemolecule104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide(Celgene), CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex),AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus),SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx),Thiazolopyrim (Dynavax), auranofin (Ridaura) (SmithKline BeechamPharmaceuticals), quinacrine (mepacrine dichlorohydrate), tenidap(Enablex), Melanin (Large Scale Biological), and anti-p38 MAPK agents byUriach.

Nucleic acid molecules encoding proteins, polypeptides, or peptides withTNF-α antagonist activity or proteins, polypeptides, or peptides withTNF-α antagonist activity can be administered to a subject with aninflammatory or autoimmune disease in accordance with the methods of theinvention. Further, nucleic acid molecules encoding derivatives,analogs, fragments or variants of proteins, polypeptides, or peptideswith TNF-α antagonist activity, or derivatives, analogs, fragments orvariants of proteins, polypeptides, or peptides with TNF-α antagonistactivity can be administered to a subject with an inflammatory orautoimmune disease in accordance with the methods of the invention. Suchderivatives, analogs, variants and fragments retain the TNF-α antagonistactivity of the full-length wild-type protein, polypeptide, or peptide.

Proteins, polypeptides, or peptides that can be used as TNF-αantagonists can be produced by any technique well-known in the art ordescribed herein. Proteins, polypeptides or peptides with TNF-αantagonist activity can be engineered so as to increase the in vivohalf-life of such proteins, polypeptides, or peptides utilizingtechniques well-known in the art or described herein. Preferably, agentsthat are commercially available and known to function as TNF-αantagonists are used in the compositions and methods of the invention.The TNF-α antagonist activity of an agent can be determined in vitroand/or in vivo by any technique well-known to one skilled in the art.

In one embodiment the method or use of the invention may compriseadministration of an antibody that is an antagonist of the biologicalactivity of Angiopoietin-2, and/or Tie-2 as an immuno-conjugate with anyone of the agents listed above.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, forexample, but not limited to, a pharmaceutical composition, containingone or more antibodies of the present invention, formulated togetherwith a pharmaceutically acceptable carrier. Such compositions mayinclude one or a combination of, for example, but not limited to two ormore different antibodies of the invention. For example, apharmaceutical composition of the invention may comprise a combinationof antibodies that bind to different epitopes on the target antigen orthat have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, such as, combined with other agents. For example,the combination therapy can include an antibody of the present inventioncombined with at least one other therapy wherein the therapy may besurgery, immunotherapy, chemotherapy, radiation treatment, or drugtherapy.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will besuitable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, selected methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

In one embodiment the compositions of the invention are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released when the microorganisms are broken downor die. Pyrogenic substances also include fever-inducing, thermostablesubstances (glycoproteins) from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Due to the potential harmfuleffects, it is advantageous to remove even low amounts of endotoxinsfrom intravenously administered pharmaceutical drug solutions. The Food& Drug Administration (“FDA”) has set an upper limit of 5 endotoxinunits (EU) per dose per kilogram body weight in a single one hour periodfor intravenous drug applications (The United States PharmacopeialConvention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeuticproteins are administered in amounts of several hundred or thousandmilligrams per kilogram body weight it is advantageous to remove eventrace amounts of endotoxin. In one embodiment, endotoxin and pyrogenlevels in the composition are less then 10 EU/mg, or less then 5 EU/mg,or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg,or less then 0.001 EU/mg. In another embodiment, endotoxin and pyrogenlevels in the composition are less then about 10 EU/mg, or less thenabout 5 EU/mg, or less then about 1 EU/mg, or less then about 0.1 EU/mg,or less then about 0.01 EU/mg, or less then about 0.001 EU/mg.

In one embodiment, the invention comprises administering a compositionwherein said administration is oral, parenteral, intramuscular,intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal,intravenous, cutaneous, subcutaneous or transdermal.

In another embodiment the invention further comprises administering acomposition in combination with other therapies, such as surgery,chemotherapy, hormonal therapy, biological therapy, immunotherapy orradiation therapy.

In another embodiment, the invention comprises administering acomposition comprising an antibody of the invention in combination withan antagonist of the biological activity of CSF1R, and/or CSF1,simultaneously, serially or separately.

In another embodiment, the invention comprises administering acomposition comprising an antibody of the invention in combination withan antagonist of the biological activity of VEGF, and/or VEGFR,simultaneously, serially or separately.

In another embodiment, the invention comprises administering acomposition comprising an antibody of the invention in combination withan antagonist of the biological activity of TNF-α, simultaneously,serially or separately.

Dosing/Administration

To prepare pharmaceutical or sterile compositions including an antibodyof the invention, the antibody is mixed with a pharmaceuticallyacceptable carrier or excipient. Formulations of therapeutic anddiagnostic agents can be prepared by mixing with physiologicallyacceptable carriers, excipients, or stabilizers in the form of, e.g.,lyophilized powders, slurries, aqueous solutions, lotions, orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak(1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y.; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, etal. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) NewEngl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl. J.Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med. 348:24-32;Lipsky, et al. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Compositions comprising antibodies of the invention can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, or by inhalation. A specific dose protocol is oneinvolving the maximal dose or dose frequency that avoids significantundesirable side effects. A total weekly dose may be at least 0.05 μg/kgbody weight, at least 0.2 μg/kg, at least 0.5 μg/kg, at least 1 μg/kg,at least 10 μg/kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 1.0mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or atleast 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu,et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al.(20003) Cancer Immunol. Immunother. 52:133-144). The dose may be atleast 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, atleast 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg.The doses administered to a subject may number at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12, or more.

For antibodies of the invention, the dosage administered to a patientmay be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Thedosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's bodyweight.

The dosage of the antibodies of the invention may be calculated usingthe patient's weight in kilograms (kg) multiplied by the dose to beadministered in mg/kg. The dosage of the antibodies of the invention maybe 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg orless, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg or less, 75 μg/kg orless, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg or less, 55 μg/kg orless, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg or less, 35 μg/kg orless, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg or less, 15 μg/kg orless, 10 μg/kg or less, 5 μg/kg or less, 2.5 μg/kg or less, 2 μg/kg orless, 1.5 μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or less, or 0.5μg/kg or less of a patient's body weight.

Unit dose of the antibodies of the invention may be 0.1 mg to 20 mg, 0.1mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg,0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 m g, 0.25 mg to5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5mg.

The dosage of the antibodies of the invention may achieve a serum titerof at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, atleast 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, atleast 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350μg/ml, at least 375 μg/ml, or at least 400 μg/ml in a subject.Alternatively, the dosage of the antibodies of the invention may achievea serum titer of at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1μg/ml, at least, 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, atleast 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, atleast 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml inthe subject.

Doses of antibodies of the invention may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001)Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al. (1983) Biopolymers 22:547-556; Langer,et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem.Tech. 12:98-105; Epstein, et al. (1985) Proc. Natl. Acad. Sci. USA82:3688-3692; Hwang, et al. (1980) Proc. Natl. Acad. Sci. USA77:4030-4034; U.S. Pat. Nos. 6,350466 and 6,316,024).

Where necessary, the composition may also include a solubilizing agentand a local anesthetic such as lidocaine to ease pain at the site of theinjection. In addition, pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985, 320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entirety. In one embodiment, an antibody, combinationtherapy, or a composition of the invention is administered usingAlkermes AIRTM pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

A composition of the present invention may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Parenteraladministration may represent modes of administration other than enteraland topical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. Alternatively, a composition of the invention can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

If the antibodies of the invention are administered in a controlledrelease or sustained release system, a pump may be used to achievecontrolled or sustained release (see Langer, supra; Sefton, 1987,CRCCrit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507;Saudek et al., 1989, N. Engl. J. Med. 321:574). Polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105); U.S. Pat. No. 5,679,377; U.S. Pat. No.5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat.No.

5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO99/20253. Examples of polymers used in sustained release formulationsinclude, but are not limited to, poly(2-hydroxy ethyl methacrylate),poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinylacetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In one embodiment, the polymer used in asustained release formulation is inert, free of leachable impurities,stable on storage, sterile, and biodegradable. A controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore antibodies of the invention. See, e.g., U.S. Pat. No. 4,526,938,PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al.,1996, “Intratumoral Radioimmunotheraphy of a Human Colon CancerXenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentirety.

If the antibody of the invention is administered topically, it can beformulated in the form of an ointment, cream, transdermal patch, lotion,gel, shampoo, spray, aerosol, solution, emulsion, or other formwell-known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms,19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topicaldosage forms, viscous to semi-solid or solid forms comprising a carrieror one or more excipients compatible with topical application and havinga dynamic viscosity, in some instances, greater than water are typicallyemployed. Suitable formulations include, without limitation, solutions,suspensions, emulsions, creams, ointments, powders, liniments, salves,and the like, which are, if desired, sterilized or mixed with auxiliaryagents (e.g., preservatives, stabilizers, wetting agents, buffers, orsalts) for influencing various properties, such as, for example, osmoticpressure. Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising antibodies are administered intranasally,it can be formulated in an aerosol form, spray, mist or in the form ofdrops. In particular, prophylactic or therapeutic agents for useaccording to the present invention can be conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice:A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic may decreasethe symptoms by at least 10%; by at least 20%; at least about 30%; atleast 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with the antibodies of the inventionmay be administered less than 5 minutes apart, less than 30 minutesapart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hoursapart, at about 2 hours to about 3 hours apart, at about 3 hours toabout 4 hours apart, at about 4 hours to about 5 hours apart, at about 5hours to about 6 hours apart, at about 6 hours to about 7 hours apart,at about 7 hours to about 8 hours apart, at about 8 hours to about 9hours apart, at about 9 hours to about 10 hours apart, at about 10 hoursto about 11 hours apart, at about 11 hours to about 12 hours apart, atabout 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hoursto 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hoursapart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hoursto 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hoursapart from the antibodies of the invention. The two or more therapiesmay be administered within one same patient visit.

The antibodies of the invention and the other therapies may becyclically administered. Cycling therapy involves the administration ofa first therapy (e.g., a first prophylactic or therapeutic agent) for aperiod of time, followed by the administration of a second therapy(e.g., a second prophylactic or therapeutic agent) for a period of time,optionally, followed by the administration of a third therapy (e.g.,prophylactic or therapeutic agent) for a period of time and so forth,and repeating this sequential administration, i.e., the cycle in orderto reduce the development of resistance to one of the therapies, toavoid or reduce the side effects of one of the therapies, and/or toimprove the efficacy of the therapies.

In certain embodiments, the antibodies of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V.V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J.Fidler (1994) Immunomethods 4:273.

The invention provides protocols for the administration ofpharmaceutical composition comprising antibodies of the invention aloneor in combination with other therapies to a subject in need thereof. Thetherapies (e.g., prophylactic or therapeutic agents) of the combinationtherapies of the present invention can be administered concomitantly orsequentially to a subject. The therapy (e.g., prophylactic ortherapeutic agents) of the combination therapies of the presentinvention can also be cyclically administered. Cycling therapy involvesthe administration of a first therapy (e.g., a first prophylactic ortherapeutic agent) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agent)for a period of time and repeating this sequential administration, i.e.,the cycle, in order to reduce the development of resistance to one ofthe therapies (e.g., agents) to avoid or reduce the side effects of oneof the therapies (e.g., agents), and/or to improve, the efficacy of thetherapies.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the invention can be administered to a subjectconcurrently. The term “concurrently” is not limited to theadministration of therapies (e.g., prophylactic or therapeutic agents)at exactly the same time, but rather it is meant that a pharmaceuticalcomposition comprising antibodies of the invention are administered to asubject in a sequence and within a time interval such that theantibodies of the invention can act together with the other therapy(ies)to provide an increased benefit than if they were administeredotherwise. For example, each therapy may be administered to a subject atthe same time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapy can be administered toa subject separately, in any appropriate form and by any suitable route.In various embodiments, the therapies (e.g., prophylactic or therapeuticagents) are administered to a subject less than 15 minutes, less than 30minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hourto about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 weekapart. In other embodiments, two or more therapies (e.g., prophylacticor therapeutic agents) are administered to a within the same patientvisit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

Another embodiment of the invention includes an assay kit for detectingAngiopoietin-1 and/or Angiopoietin-2 in mammalian tissues, cells, orbody fluids to screen for angiogenesis-related diseases. The kitincludes an antibody that binds to Angiopoietin-1 and/or Angiopoietin-1and a means for indicating the reaction of the antibody withAngiopoietin-1 and/or Angiopoietin-2, if present. The antibody may be amonoclonal antibody. In one embodiment, the antibody that bindsAngiopoietin-2 is labeled. In another embodiment the antibody is anunlabeled primary antibody and the kit further includes a means fordetecting the primary antibody. In one embodiment, the means includes alabeled second antibody that is an anti-immunoglobulin. In someembodiments, the antibody is labeled with a marker selected from thegroup consisting of a fluorochrome, an enzyme, a radionuclide and aradio-opaque material.

Incorporation By Reference

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. In addition, the following U.S. provisionalpatent applications 61/023,958 filed Jan. 28, 2008, 61/100,063 filedSep. 25, 2008, and 61/142,778 filed Jan. 6, 2009 are hereby incorporatedby reference herein in their entireties for all purposes.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples detail certain embodiments of the invention anddescribes the best mode contemplated by the inventors. It will beappreciated, however, that no matter how detailed the foregoing mayappear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

Specific Embodiments:

-   -   1. An isolated antibody that binds to Ang-2, wherein said        antibody comprises a variable light chain, said light chain        comprising a sequence selected from the group consisting of SEQ        ID No:3 (MEDI1); SEQ ID No:4 (MEDI2); SEQ ID No:5 (MEDI3); SEQ        ID No:6 (MEDI4); and SEQ ID No:8 (MEDI6).    -   2. The antibody of embodiment 1, wherein said antibody is an        IgG1 or an IgG2 isotype antibody.    -   3. The antibody of embodiment 1 or 2, wherein said antibody        further comprises a variable heavy chain region comprising SEQ        ID No:7 (MEDI5).    -   4. The antibody of any of embodiments 1-3, wherein said        antibody, when produced, exhibits a production efficiency in a        mammalian host cell equal to or greater than 2 times the        production efficiency of the Ang-2 antibody 3.19.3.    -   5. The antibody of embodiment 4, wherein said production        efficiency is equal to or greater than 3 times the production        efficiency of the Ang-2 antibody 3.19.3.    -   6. The antibody of embodiment 4, wherein said production        efficiency is equal to or greater than 5 times the production        efficiency of the Ang-2 antibody 3.19.3.    -   7. A nucleic acid encoding the antibody of any of embodiments        1-6.    -   8. A vector comprising the nucleic acid of embodiment 7    -   9. A host cell comprising the vector of embodiment 8.    -   10. A pharmaceutical composition comprising the antibody of any        of embodiments 1-6 and an excipient.    -   11. A method of preventing, treating, or managing cancer in an        animal in need thereof, said method comprising administering to        said animal a dose of an effective amount of the composition of        embodiment 10.    -   12. A method of preventing metastasis of cancer in an animal in        need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   13. A method of preventing recurrences of cancer in an animal in        need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   14. A method of preventing advancement of cancer in an animal in        need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   15. A method of preventing development of cancer from a        pre-cancerous state in an animal in need thereof, said method        comprising administering to said animal an effective amount of        the composition of embodiment 10.    -   16. A method of preventing symptoms of cancer in an animal in        need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   17. A method of promoting tumor regression of a cancer in an        animal in need thereof, said method comprising administering to        said animal a dose of an effective amount of the composition of        embodiment 10.    -   18. A method of inhibiting tumor cell proliferation in an animal        in need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   19. A method of depleting malignant tumor cells in an animal in        need thereof, said method comprising administering to said        animal a dose of an effective amount of the composition of        embodiment 10.    -   20. A method of inhibiting angiogenesis of a cancer tumor in an        animal in need thereof, said method comprising administering to        said animal a dose of an effective amount of the composition of        embodiment 10.    -   21. The method of any of embodiments 11-20, wherein said method        comprises an additional dosing to said animal of one or more        other cancer therapies.    -   22. The method of embodiment 21, wherein said one or more other        cancer therapies are chemotherapies, biological        therapies/immunotherapies, radiation therapies, hormonal        therapies, or surgery.    -   23. The method of any of embodiments 11-22, wherein said method        further comprises the administration of another therapeutic        agent that is not a cancer therapeutic agent.    -   24. The method of embodiment 23, wherein said therapeutic agent        is an anti-emetic agent, anti-fungal agent, anti-parasitic        agent, anti-inflammatory agent, immunomodulatory agent,        anti-viral agent, or antibiotic.    -   25. The method of any of embodiments 21-24, wherein said        chemotherapy is selected from the group consisting of        5-Flurouracil, carboplatin, and paclitaxel.    -   26. The method of any of embodiments 21-25, wherein said        immunotherapy is the administration of bevacizumab or an        antibody that competes for the same epitope as bevacizumab.    -   27. The method of any of embodiments 11-26, wherein said cancer        or tumor is selected from the group consisting of melanoma,        colon, colorectal, lung, small cell lung carcinoma, non-small        cell lung carcinoma, breast, rectum, stomach, glioma, prostate,        ovary, testes, thyroid, blood, kidney, liver, hepatocellular        carcinoma pancreas, brain, neck, glioblastoma, endometrial        cancer, and central nervous system cancer.    -   28. The method of any of embodiments 11-27, wherein said animal        has been previously treated by administration of one or more        cancer therapies but not by administration of the composition of        embodiment 10.    -   29. The method of any embodiments 11-27, wherein said animal has        been previously treated with chemotherapy alone, or in        combination with one or more radiation therapies,        biological/immunotherapies, hormonal therapies or surgery.    -   30. The method of any of embodiments 11-27, wherein said animal        has been previously treated with radiation therapy alone, or in        combination with one or more chemotherapies, biological        therapies/immunotherapies, hormonal therapies or surgery.    -   31. The method of any of embodiments 11-27, wherein said animal        has been previously treated with biological        therapies/immunotherapies alone, or in combination with one or        more chemotherapies, radiation therapy, hormonal therapies or        surgery.    -   32. The method of any of embodiments 11-27, wherein said animal        has been previously treated with hormonal therapies alone, or in        combination with one or more chemotherapies, radiation therapy,        biological therapies/immunotherapies or surgery.    -   33. The method of any of embodiments 11-27, wherein said animal        has been previously treated with surgery alone, or in        combination with one or more chemotherapies, radiation therapy,        hormonal therapies or biological therapies/immunotherapies.    -   34. The method of any of embodiments 11-33, wherein said cancer        is refractory to chemotherapy or radiation therapy.    -   35. The method of any of embodiments 11-34, wherein said        administration is intravenously, subcutaneously, intratumorally,        intramuscularly, parenterally, or orally.    -   36. The method of any of embodiments 21-35, wherein said        composition and cancer therapy are administered by the same mode        of administration.    -   37. The method of any of embodiments 21-35, wherein said        composition and cancer therapy are administered by a different        mode of administration.    -   38. The method of any of embodiments 21-35, wherein said        composition and cancer therapy are administered in the same        dosage form.    -   39. The method of any of embodiments 21-35, wherein said        composition and cancer therapy are administered in different        dosage forms.    -   40. The method of any of embodiments 21-39, wherein said cancer        therapy is selected from the group consisting of radiation        therapies, biological therapies/immunotherapies, hormonal        therapies and surgery.    -   41. A method of preventing, treating, or managing        disease-related angiogenesis in an animal in need thereof, said        method comprising administering to said animal a dose of an        effective amount of the composition of embodiment 10.    -   42. The method of embodiment 41, wherein the disease-related        angiogenesis is associated with seronegative arthritis,        seropositive arthritis, arthritis related to other        arthropathies, osteoarthritis or SLE.    -   43. The method of embodiment 42, wherein the seropositive        arthritis is rheumatoid arthritis.    -   44. A method of preventing recurrences of disease-related        angiogenesis in an animal in need thereof, said method        comprising administering to said animal a dose of an effective        amount of the composition of embodiment 10.    -   45. A method of preventing advancement of disease-related        angiogenesis in an animal in need thereof, said method        comprising administering to said animal a dose of an effective        amount of the composition of embodiment 10.    -   46. A method of treating rheumatoid arthritis in an animal in        need thereof, said method comprising administering to said        animal an effective amount of the composition of embodiment 10.    -   47. A method of preventing symptoms of disease-related        angiogenesis in an animal in need thereof, said method        comprising administering to said animal a dose of an effective        amount of the composition of embodiment 10.    -   48. The method of any of embodiments 41-47, wherein said method        comprises an additional dosing to said animal of one or more        other anti-inflammatory therapies.    -   49. The method of embodiment 48, wherein said one or more other        anti-inflammatory therapies are chemotherapies, biological        therapies/immunotherapies, radiation therapies, hormonal        therapies, or surgery.    -   50. The method of embodiment 49, wherein said biological        therapy/immunotherapy is a TNF-α antagonist.    -   51. The method of embodiment 50, wherein said TNF-α is selected        from etanercept(ENBREL®), adalimumab(HUMIRA®), and        infliximab(REMICADE®).    -   52. The method of any of embodiments 41-51, wherein said method        further comprises the administration of another therapeutic        agent that is not an anti-inflammatory therapeutic agent.    -   53. The method of embodiment 52, wherein said therapeutic agent        is an anti-emetic agent, anti-fungal agent, anti-parasitic        agent, anti-cancer agent, immunomodulatory agent, anti-viral        agent, or antibiotic.    -   54. The method of any of embodiments 41-53, wherein said animal        has been previously treated by administration of one or more        anti-inflammatory therapies but not by administration of the        composition of embodiment 10.    -   55. The method of any of embodiments 41-53, wherein said animal        has been previously treated with chemotherapy alone, or in        combination with one or more radiation therapies,        biological/immunotherapies, hormonal therapies or surgery.    -   56. The method of any of embodiments 41-53, wherein said animal        has been previously treated with radiation therapy alone, or in        combination with one or more chemotherapies, biological        therapies/immunotherapies, hormonal therapies or surgery.    -   57. The method of any of embodiments 41-53, wherein said animal        has been previously treated with biological        therapies/immunotherapies alone, or in combination with one or        more chemotherapies, radiation therapy, hormonal therapies or        surgery.    -   58. The method of any of embodiments 41-53, wherein said animal        has been previously treated with hormonal therapies alone, or in        combination with one or more chemotherapies, radiation therapy,        biological therapies/immunotherapies or surgery.    -   59. The method of any of embodiments 41-53, wherein said animal        has been previously treated with surgery alone, or in        combination with one or more chemotherapies, radiation therapy,        hormonal therapies or biological therapies/immunotherapies.    -   60. A method of reducing endothelial cell proliferation in an        animal, said method comprising administration of a dose of an        effective amount of the composition of embodiment 10.    -   61. A method of inhibiting Ang-2 and/or Ang-1 binding to Tie-2        in an animal, said method comprising administration of a dose of        an effective amount of the composition of embodiment 10.    -   62. A method of inhibiting Tie-2 phosphorylation in an animal,        said method comprising administration of a dose of an effective        amount of the composition of embodiment 10.    -   63. A method of reducing levels of circulating Ang-2 and/or        Ang-1 polypeptide in an animal, said method comprising        administration of a dose of an effective amount of the        composition of embodiment 10.    -   64. A pharmaceutical composition comprising a combination of i)        an antagonist of the biological activity of Angiopoietin-2        and/or Tie-2, and ii) an antagonist of the biological activity        of CSF1R, and/or CSF1.    -   65. The composition according to embodiment 64, wherein the        antagonist of Angiopoietin-2 is an antibody.    -   66. The composition according to embodiment 65, wherein the        antagonist of Angiopoietin-2 is a fully human monoclonal        antibody.    -   67. The composition according to embodiments 65 or 66, wherein        the antibody binds to the same epitope as any one of fully human        monoclonal antibodies selected from the group consisting of        3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3,        5.28.1, 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and MEDI4/5.    -   68. The composition according to embodiment 65, wherein the        antibody is a fully human monoclonal antibody selected from the        group consisting of: 33.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,        5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5,        MEDI3/5, MEDI6/5, and MEDI4/5.    -   69. The composition according to any of embodiments 64-68,        wherein the antagonist of the biological activity of CSF1R is a        tyrosine kinase inhibitor.    -   70. The composition according to embodiment 69, wherein the        antagonist of the biological activity of CSF1R is selected from        any one of:    -   2-chloro-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-benzamide;    -   2-methyl-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-pyridin-3-ylbenzamide;    -   2-chloro-5-[(3-cyclopropylbenzoyl)amino]-N-pyridin-3-ylbenzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-pyridin-3-ylbenzamide;    -   5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-[(3-chlorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;    -   5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-N-1,3-thiazol-5-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;    -   2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;    -   5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-1,3-thiazol-5-ylbenzamide;    -   2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-[(3-chlorobenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-chloro-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;    -   2-chloro-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methylbenzoyl)amino]-N-methyl-1,3-thiazole-2-carboxamide;    -   5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-chlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[3,4-dichlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3-cyclopropylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   5-[(3,5-dimethylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-methyl-5-[(3-methylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2,6-dichloro-N-(4-methyl-3-{[(2-methyl-1,3-thiazol-5-yl)amino]carbonyl}phenyl)isonicotinamide;    -   2-methyl-5-{[(3-methylcyclohexyl)carbonyl]amino}-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-(pentanoylamino)benzamide;    -   2-methyl-5-[(4-methylhexanoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2,3-dichlorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(3-chloro-2-fluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-5-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-7-(2-methoxyethoxy)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-isopropylpiperidin-4-yl)quinoline-3-carboxamide;    -   7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoline-3-carboxamide;    -   4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;    -   4-[(2,4-difluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;        and    -   4-[(3-chloro-2-fluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquinoline-3-carboxamide;    -   7-Ethoxy-4-[(2-fluoro-4-methyl-phenyl)amino]-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   4-(2-Fluoro-4-methylphenylamino)-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   4-[(2,4-Difluorophenyl)amino]-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoline-3-carboxamide;    -   6-[(3R,5        S)-3,5-Dimethylpiperazin-1-yl]-4-[(2-fluoro-4-methylphenyl)amino]-7-methoxycinnoline-3-carboxamide;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-7-methoxycinnoline-3-carboxamide;    -   7-Ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]cinnoline-3-carboxamide;    -   4-[(3-Chloro-2-fluorophenyl)amino]-6-[(3R,5 S)-3        ,5-dimethylpiperazin-1-yl]-7-methoxycinnoline-3-carboxamide;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxycinnoline-3-carboxamide        hydrochloride;    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-[1-(2-hydroxyethyl)piperidin-4-yl]-7-methoxycinnoline-3-carboxamide;        and    -   4-[(2-Fluoro-4-methylphenyl)amino]-6-{4-[(2R)-2-hydroxypropanoyl]piperazin-1-yl}-7-methoxycinnoline-3-carboxamide;    -   or a pharmaceutically acceptable salt thereof.    -   71. A pharmaceutical composition comprising a combination        according to any one of embodiments 62 to 70, in association        with a pharmaceutically-acceptable excipient or carrier.    -   72. A pharmaceutical composition according to embodiment 71, for        use in the treatment of disease-related angiogenesis or        inflammation.    -   73. A pharmaceutical composition according to embodiment 71, for        use in the treatment of cancer.

74. A method of treating disease-related angiogenesis or inflammation inan animal in need thereof with a combination according to any one ofembodiments 62-73.

-   -   75. A method of treating cancer in an animal in need thereof        with a combination according to any one of embodiments 62-73.    -   76. A composition comprising an antagonist of the biological        activity of Angiopoietin-2, and/or Tie-2; and a chemotherapeutic        agent.    -   77. The composition according to embodiment 76, wherein the        antagonist of Angiopoietin-2 is an antibody.    -   78. The composition according to embodiment 77, wherein the        antagonist of Angiopoietin-2 is a fully human monoclonal        antibody.    -   79. The composition according to any one of embodiments 77 or        78, wherein the antibody binds to the same epitope as an        antibody selected from the group consisting of 3.31.2, 5.16.3,        5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3,        MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and MEDI4/5.    -   80. The composition according to embodiment 78, wherein the        antibody is a fully human monoclonal antibody selected from the        group consisting of 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,        5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5,        MEDI3/5, MEDI6/5, and MEDI4/5.    -   81. The composition according to any of embodiments 76-80,        wherein the chemotherapeutic agent is selected from the group        consisting of docetaxel, AZD4877, vincristine, vinblastine,        vindesine and vinorelbine, taxol, taxotere, 5-fluorouracil,        gemcitabine, fluoropyrimidines tegafur, raltitrexed,        capecitabine, methotrexate, pemetrexed, cytosine arabinoside,        hydroxyurea; irinotecan, etoposide topotecan, camptothecin        teniposide, amsacrine, oxaliplatin, cisplatin oxaliplatin,        5-fluorouracil, irinotecan, gemcitabine and carboplatin.    -   82. The composition according to any of embodiments 76 to 81 in        association with a pharmaceutically acceptable excipient or        carrier.    -   83. A method of antagonizing the biological activity of        Angiopoietin-2, and/or Tie-2 comprising administering the        composition according to any of embodiments 76 to 82.    -   84. A method of producing an anti-cancer effect in a patient        comprising administering a therapeutically effective amount of a        composition of any one of embodiments 76 to 82.    -   85. A method of reducing tumor growth in an animal comprising        administering a therapeutically effective amount of a        composition of any one of embodiments 76 to 82.

VI. SEQUENCES

3.19.3 light chain SEQ ID No.: 1EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRL LI

GASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The boxed residue in this sequence represents an unpaired cysteine (C49)that may be changed to any other amino acid. Examples of such changesare highlighted in the light chain sequences below.

3.19.3 heavy chain SEQ ID No: 2 QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGMHW

RQAPGKGLEWV AVISHDGNNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGIDFWSGLNWFDPWGQGTLVTVSS

The boxed residue in this sequence represents an example of a residuethat may be “backmutated” to another residue. One example of such a“backmutation” is represented in the MEDI5 heavy chain sequence.

MEDI1 light chain SEQ ID No: 3EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLL I

GASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

MEDI2 light chain SEQ ID No: 4EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLL I

GASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

MEDI3 light chain SEQ ID No: 5EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLL I

GASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

MEDI4 light chain SEQ ID No: 6EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLL I

GASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

MEDI5 heavy chain SEQ ID No: 7 QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGMHW

RQAPGKGLEWV AVISHDGNNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGIDFWSGLNWFDPWGQGTLVTVSS

MEDI6 light chain SEQ ID No: 8EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLL I

GASSWATGI

DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

VII. EXAMPLES Example 1 Potency of Modified Ang-2 Antibodies

In this example, Ang-2 specific antibodies comprising the light andheavy chains of the Ang-2 antibody 3.19.3 have been modified such thatamino acid substitutions for cysteine at position 49 of the light chainwere introduced. The potency of the resultant antibodies was measured inan Ang-2:Tie-2 potency assay. The results are presented in Table 1below.

TABLE 1 Potency of modified Ang-2 antibodies Antibody IC50 3.19.3 (WT)0.06 C49A (MEDI4) SEQ ID No.: 6 0.03 C49D (MEDI3) SEQ ID No.: 5 0.07C49E 0.06 C49F 0.06 C49G 0.09 C49H 0.98 C49I 0.04 C49K 103.40 C49L 0.05C49M 0.05 C49N (MEDI2) SEQ ID No.: 4 0.04 C49P 0.07 C49Q 0.12 C49R 5.48C49S 0.14 C49T (MEDI1) SEQ ID No.: 3 0.04 C49V 0.08 C49W 1.02 C49Y 0.73Control Ab 0.05

Results: In an Ang-2:Tie-2 potency assay, various modifications ofposition 49 resulted in antibodies with similar potency. Exemplaryantibodies include but are not limited to C49A, C49T, C49N, and C49D.Some of the modifications that included bulky, hydrophobicsubstitutions, such as C49W resulted in lowered potency demonstrated byan increased IC50 value. Other modifications, including various chargedresidues such as C49K and C49H also resulted in lowered potencydemonstrated by an increased IC50 value.

Example 2 Improved Antibody Production Efficiency

In this example, Ang-2 specific antibodies comprising the light andheavy chains of the Ang-2 antibody 3.19.3 have been modified such thatamino acid substitutions for cysteine at position 49 of the light chainwere introduced. The relative expression of the resulting antibodies wasmeasured. In addition, position 37 of the heavy chain of severalantibodies was also modified to introduce a Val residue (MEDI5).

Materials and Methods: Vectors encoding the heavy and light chains of3.19.3 (SEQ ID Nos. 2 and 1, respectively), as well as, heavy and lightchains set forth as SEQ ID Nos. 7 and 3, respectively (in both an IgG1and IgG2 format) were expressed in 293F cells with the followingprotocol: Viable cells (>95%) were diluted in Freestyle293® media(Invitrogen) at a cell density of 1.0×10⁶ cells/min. The various DNApreparations were diluted in 293Fectin® (Invitrogen) and added to cellsas per the manufacturers directions. On Day 6 of the transformation, theexpressed antibodies were harvested by collecting the culturesupernatant. Antibody levels were measured by Protein A binding prior topurification.

Results: The production efficiencies of the “MEDI1/5 IgG1” (having theheavy and light chains set forth as SEQ ID Nos. 7 and 3 in an IgGformat), and “MEDI1/5 IgG2” (having the heavy and light chains set forthas SEQ ID Nos. 7 and 3 in an IgG format) antibodies were increased ascompared to the production efficiency of the 3.19.3 antibody. Theresults are summarized in Table 2.

TABLE 2 Improved production efficiency of Ang-2 antibodies Antibody3.19.3 MEDI1/5 IgG1 MEDI1/5 IgG2 Yield  10 mg/L 140 mg/L 260 mg/LRecovery 0.2 g  1.4 g  2.7 g

These results demonstrate that the substitution at position 49 ofcysteine for threonine in the variable light chain coupled with thesubstitution at position 37 of glycine to valine in the light chain ofan Ang-2 specific antibody leads to greatly improved productionefficiencies (i.e. yield and/or recovery).

Example 3 Increased Stability of Ang-2 Antibodies

In an attempt to assess the increased stability of the cysteinesubstituted Ang-2 antibodies, a stability study was performed. The WT3.19.3 antibody, as well as, MEDI1/5 in an IgG1 and IgG2 format wereconcentrated to 10 mg/ml in 10 mM histidine pH 6.0. Samples of theaforementioned formulations were incubated at either 25° C. or 40° C.for two weeks. As a measure of stability, the rate of aggregation (%aggregation/mth) was calculated after the two week time point. Theresults are presented in Table 3.

TABLE 3 Stability of Ang-2 antibodies Sample % Agg/mth @ 40° C. %Agg/mth @ 25° C. WT (3.19.3) 42.6 2.97 MEDI1/5 IgG1 0.21 0.21 MEDI1/5IgG2 0.82 0.61

As demonstrated in Table 2, the MEDI1/IgG1 and IgG2 antibodies exhibitan enhanced stability as compared to the WT (3.19.3) antibody. Also, asdemonstrated in FIG. 2, much of the heterogeneity demonstrated by thewild type antibody (3.19.3) was abolished in the MEDI1/5 antibodies inan IgG1 or IgG2 formats. The chromatograph in FIG. 2 demonstrates thatreplacement of C49 in the wild type antibody reduces or eliminates themultiple antibody species present in samples.

Example 4 Increased Stability of Ang-2 Antibodies

In an attempt to assess the increased stability of the cysteinesubstituted Ang-2 antibodies, a Differential Scanning calorimetry (DSC)analysis of WT (3.19.3) and MEDI1/5 IgG1 and IgG2 antibodies wasperformed. In this example, formulations of WT, MEDI1/5IgG1, and MEDI1/5IgG2 antibodies were prepared at 1 g/L antibody in 10 mM histidine, pH6.0 and subjected to DSC analysis. The results are presented in FIG. 1.As presented, the WT antibody exhibits a melting temperature of about61° C. The MEDI1/5 IgG1 antibody exhibits a higher melting temperatureof about 76° C. The MEDI1/5 IgG2 also exhibits a higher meltingtemperature of about 76° C., however it subsequently falls out ofsolution, which was observed in two independent trials (trial 1 and 2).

These results suggest that MEDI1/5 antibodies exhibit increasedstability over the WT (3.19.3) antibody as measured by meltingtemperature.

Example 5 Binding Profiles of Ang-2 Antibodies

In this example the Ang-2 antibody, 3.19.3, and a modified 3.19.3antibody comprising a C49T (MEDI1/5) substitution were analyzed forAng-2 binding in a competitive ELISA format assay.

Materials and Methods:

Competitive Tie-2 Fc/Ang2 ELISA: Maxisorp ELISA plates (Nunc, Rochester,N.Y.) were coated with 100 μl of 4 μg/ml Tie-2 Fc (R & D Systems,Minneapolis, Minn.) in 0.1M carbonate buffer pH 9.4 (Pierce, Rockford,Ill.) and incubated overnight at 4° C. The following day, plates wereblocked for 1 hour at room temperature with 200 μl of phosphate bufferedsaline (PBS) (Invitrogen, Carlsbad, Calif.) containing 0.5% bovine serumalbumin (BSA) (Sigma, St. Louis, Mo.) and 0.1% Tween-20 (Sigma, St.Louis, Mo.). Plates were washed 3 times with wash buffer (PBS containing0.05% Tween-20). 50 μl of 11-point serial tertiary dilutions (30 μg/mlhigh concentration) of 3.19.3 or modified anti-Ang2 antibody wereplated, with PBS as a negative control. For antibody capture, 50 μl of200 ng/ml Biotin Ang2 (R & D Systems, Minneapolis, Minn.) were added andplates were incubated for 2 hours at room temperature. Plates werewashed 3 times with wash buffer. Following the wash, 100 ul of 1:5000streptavidin HRP (Pierce, Rockford, Ill.) dilution in wash buffer wereadded and incubated for 1 hour at room temperature. Plates were washed 3times with wash buffer. Plates were developed by adding 100 μl of TMBperoxidase substrate (KPL, Gaithersburg, Md.) for 5 minutes. Thereaction was stopped by adding 100 μl/well of 1M phosphoric acid.Optical densities were measured at 450 nm with a microplate reader(Molecular Devices, Sunnyvale, Calif.).

Results: As presented in FIG. 3, the ELISA results demonstrate that theMEDI1/5 antibody in either an IgG1 or an IgG2 format exhibit a verysimilar binding profile for Ang-2 as compared to the 3.19.3 antibody asmeasured by competition for Tie-2 in an ELISA format.

Example 6 Combination Studies

The in vivo efficacy of 3.19.3 in combination with small molecule CSF1Rtyrosine kinase inhibitors has been evaluated.

Study 6.1 Determination of the Therapeutic Efficacy of mAb 3.19.3 inCombination with the CSF1R Antagonist AZD6495 in a MCF7 Breast CancerXenograft Model

The anti-tumor activity of the anti-Angiopoietin-2 monoclonal antibody3.19.3 in combination with the small molecule CSF1R tyrosine kinaseinhibitor AZD6495(4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide)was evaluated in a xenograft model of human breast carcinoma using theMCF7 cell line (FIG. 4 a).

Materials and Methods: Breast carcinoma MCF7 cells were cultured inflasks as routine until the cells reach sub-confluence. Immunodeficient7-10 week old male NCr-nude mice were subcutaneously implanted with8×10⁶ MCF7 cells suspended 1:1 in Matrigel in the right flank.17-B-Estradiol pellets (0.72 mg/pellet) were also implanted as standardprocedure to support the growth of this ER positive (estrogen requiring)cell line.

The mice were then randomized into cohorts containing 15 mice once thetumors reached approximately 100 mm³. The mice were treated byintraperitoneally (IP) injection with mAb 3.19.3 (10 mg/kg) twice perweek for 3 weeks thereafter, or by oral administration of CSF1Rinhibitor A(AZD6495) at (30 mg/kg) following bid dosing for 18 days. Forall experiments, 0.5% HPMC was used as an oral vehicle only control. Thebody weights of each animal, and the dimensions of each tumor weremeasured twice per week. The volume of the tumor was calculated as:Volume=Length×(Width)²×0.5 cm³, or by bilateral Vernier calipermeasurement and, taking length to be the longest diameter across thetumor and width the corresponding perpendicular, calculated using theformula (π/6)×(length×width)×√(length×width). Growth inhibition from thestart of treatment was assessed by comparison of the differences intumor volume between control and treated groups. The summary studydesign was as follows:

TABLE 4 Study design Group Treatment # Mice Schedule Route A Veh (0.5%HPMC) 15 bid × 18 d p.o. B mAb3.19.3, 10 mg/kg 15 q3.5 d × 3 wk i.p. CAZD6495, 30 mg/kg 15 bid × 18 d p.o. D mAb3.19.3, 10 mg/kg + 15 q3.5 d ×3 wk i.p. AZD6495, 30 mg/kg bid × 18 d p.o.

As illustrated in FIG. 4 a, 3.19.3 and AZD6495 delayed the growth of theMCF7 tumors as single agents. However the combination of 3.19.3 andAZD6495 had a greater effect than the single agents alone as illustratedin FIG. 4 a. The % tumor growth inhibition achieved is as follows:

3.19.3 (10 mg/kg 2×wk)=62% inhibition; (p<0.04)

AZD6495 (30 mg/kg bid)=32% inhibition; (p<0.42)

-   -   Combination 3.19.3+AZD6495=81% inhibition (p<0.01)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 4 b). Changes in macrophage populations are being measured viaF4/80 staining and Fluorescence Activated Cell Sorting and the tumortissue analyzed via CD31+ vessel staining density to examine any effectson the tumor associated vasculature. CD31 staining density can bemeasured by threshold and by manual grid counting methods. Thecombination of 3.19.3 with CSF1R tyrosine kinase inhibitors is expectedto produce a significantly greater effect on both tumor associatedmacrophage populations and CD31 staining blood vessels.

These results demonstrate that combination treatment with the anti-Ang2antibody 3.19.3 and the small molecule CSF1R antagonist, AZD6495, leadsto improvements in efficacy without additive toxicity in a pre-clinicalmodel of breast cancer.

Study 6.2. Determination of the Therapeutic Efficacy of mAb 3.19.3 inCombination with the CSF1R Antagonist AZD6495 in a MDA-MB-231 OrthotopicBreast Cancer Xenograft Model

The anti-tumor activity of the anti-Angiopoietin-2 monoclonal antibody3.19.3 in combination with the small molecule CSF1R tyrosine kinaseinhibitor AZD6495 was evaluated in a xenograft model of human breastcarcinoma using the MDA-MB-231 cell line (FIG. 5 a).

Breast adenocarcinoma MDA-MB-231 cells were cultured in flasks asroutine until the cells reach sub-confluence. Immunodeficient 7-10 weekold female nude mice were orthotopically implanted with 8×10⁶ MDA-MB-231cells suspended 1:1 in Matrigel into the mammary fat pad. The mice werethen randomized into cohorts containing 10 mice once the tumors reachedapproximately 100 mm³. The mice were treated by intraperitoneally (IP)injection with mAb 3.19.3 (10 mg/kg) twice per week for 3 weeksthereafter, or by oral administration of AZD6495 (30 mg/kg) followingbid dosing for 18 days. For all experiments, 0.5% HPMC was used as anoral vehicle only control. The body weights of each animal, and thedimensions of each tumor were measured twice per week. The volume of thetumor was calculated as: Volume=Length×(Width)²×0.5 cm³, or by bilateralVernier caliper measurement and, taking length to be the longestdiameter across the tumor and width the corresponding perpendicular,calculated using the formula (π/6)×(length×width)×√(length×width).Growth inhibition from the start of treatment was assessed by comparisonof the differences in tumor volume between control and treated groups.

The summary study design was as follows;

TABLE 5 Study design Group Treatment # Mice Schedule Route A Veh (0.5%HPMC) 10 bid × 18 d p.o. B mAb3.19.3, 10 mg/kg 10 q3.5 d × 3 wk i.p. CAZD6495, 30 mg/kg 10 bid × 18 d p.o. D mAb3.19.3, 10 mg/kg + 10 q3.5 d ×3 wk i.p. AZD6495, 30 mg/kg bid × 18 d p.o.

As illustrated in FIG. 5 a, 3.19.3 and AZD6495 delayed the growth of theMDA-MB-231 tumors as single agents. However the combination of 3.19.3and AZD6495 had a greater effect than the single agents alone asillustrated in FIG. 5 a. The % tumor growth inhibition achieved is asfollows:

3.19.3 (10 mg/kg 2×wk)=17% inhibition; (p<0.20)

AZD6495 (30 mg/kg bid)=25% inhibition; (p<0.04)

Combination 3.19.3+AZD6495=52% inhibition (p<0.09)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 5 b). Changes in macrophage populations are being measured viaF4/80 staining and Fluorescence Activated Cell Sorting and the tumortissue analyzed via CD31+ vessel staining density to examine any effectson the tumor associated vasculature. CD31 staining density can bemeasured by threshold and by manual grid counting methods. Thecombination of 3.19.3 with CSF1R tyrosine kinase inhibitors is expectedto produce a significantly greater effect on both tumor associatedmacrophage populations and CD31 staining blood vessels.

These results demonstrate that combination treatment with the anti-Ang2antibody 3.19.3 and the small molecule CSF1R antagonist AZD6495 leads toimprovements in efficacy without additive toxicity in a pre-clinicalmodel of breast cancer.

Example 7 Combination Studies

The activity of the monoclonal antibody 3.19.3 was evaluated incombination studies with chemotherapeutic agents to determine the invivo efficacy and tolerability in human tumor xenograft models.

Study 7.1. Determination of the Therapeutic efficacy of MonoclonalAntibody 3.19.3 in Combination with 5-Flurouracil in LoVo XenograftTumors

The anti-tumor activity of 3.19.3 was evaluated in combination with5-Fluoruracil (5FU) in the LoVo xenograft model of colorectal cancer.LoVo cells were cultured in flasks as routine until the cells reachedsub-confluence. Cell suspensions containing approximately 3×10E6 cellswere injected subcutaneously into the flank of female Swiss nude mice.When the tumor volume reached 200 mm³, the mice were randomized intreatment groups of 8-10 mice and the treatments were initiated. 3.19.3(10 mg/kg) in saline was injected intraperitoneally, twice per week for2 weeks and 5-Flurouracil (100 mg/kg) was administered byintraperitoneal administration following a weekly schedule. Thedimensions of each tumor and body weights were measured at least twiceper week. The volume of the tumor was calculated as:Volume=Length×(Width)×0.5 (cm³). As illustrated in FIG. 6 a, 3.19.3 and5FU delayed the growth of the LoVo tumors as single agent. However thecombination of 3.19.3 and 5FU had a greater effect than the singleagents alone as illustrated in FIG. 6 a. The % tumor growth inhibitionachieved is as follows:

3.19.3 (10 mg/kg 2×wk)=59% inhibition; (p<0.09)

5 FU (100 mg/kg/week)=62% inhibition; (p<0.02)

Combination 3.19.3+5FU=85% inhibition (p<0.007)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 6 b). These results demonstrate that combination treatment withthe anti-Ang2 antibody 3.19.3 and 5-flurouracil leads to improvements inefficacy without additive toxicity in a pre-clinical model of coloncancer, providing the basis for further clinical investigation of thiscombination.

Study 7.2. Determination of the Therapeutic efficacy of MonoclonalAntibody 3.19.3 in Combination with Irinotecan in HT-29 Xenograft Tumors

The anti-tumor activity of 3.19.3 was evaluated in combination withIrinotecan in the HT-29 xenograft model of colorectal cancer. HT-29cells were cultured in flasks as routine until the cells reachedsub-confluence. Cell suspensions containing approximately 3×10E6 cellswere injected subcutaneously into the flank of female Swiss nude mice.When the tumor volume reached 200 mm³, the mice were randomized intreatment groups of 8-10 mice and the treatments were initiated. 3.19.3(10 mg/kg) in saline was injected intraperitoneally, twice per week for2 weeks. Irinotecan (35 mg/kg) was administered by intravenousadministration following a weekly schedule. The dimensions of each tumorand body weights were measured at least twice per week. The volume ofthe tumor was calculated as: Volume=Length×(Width)×0.5 (cm³). Asillustrated in FIG. 7 a, 3.19.3 and Irinotecan delayed the growth of theHT29 tumors as single agents. However the combination of 3.19.3 andIrinotecan had a greater effect than the single agents alone asillustrated in FIG. 7 a. The % tumor growth inhibition achieved is asfollows:

3.19.3 (10 mg/kg 2×wk)=44% inhibition; (p<0.005)

Irinotecan (35 mg/kg/week)=56% inhibition; (p<0.006)

Combination 3.19.3+Irinotecan=71% inhibition (p<0.0001)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 7 b). These results demonstrate that combination treatment withanti-Ang2 antibody 3.19.3 and Irinotecan leads to improvements inefficacy without additive toxicity in a pre-clinical model of coloncancer, and providing the basis for further clinical investigation ofthis combination.

Study 7.3. Determination of the therapeutic efficacy of monoclonalantibody 3.19.3 in combination with Gemcitabine in Colo205 xenografttumors

The anti-tumor activity of 3.19.3 was evaluated in combination withGemcitabine in the Colo205 xenograft model of colorectal cancer. Colo205cells were cultured in flasks as routine until the cells reachedsub-confluence. Cell suspensions containing approximately 3×10E6 cellswere injected subcutaneously into the flank of female Swiss nude mice.When the tumor volume reached 200 mm³, the mice were randomized intreatment groups of 8-10 mice and the treatments were initiated. 3.19.3(10 mg/kg) in saline was injected intraperitoneally, twice per week for2 weeks. Gemcitabine (50 mg/kg) was administered by intravenousadministration following a q3d schedule. The dimensions of each tumorand body weights were measured at least twice per week. The volume ofthe tumor was calculated as: Volume=Length×(Width)×0.5 (cm³). Asillustrated in FIG. 8 a, 3.19.3 delayed the growth of the Colo205 tumorsas single agent however the Colo205 tumors were fairly refractory toGemcitabine treatment resulting in a modest 8% tumor growth delay.However the combination of 3.19.3 and Gemcitabine had a greater effectthan the single agents alone as illustrated in FIG. 8 a. The % tumorgrowth inhibition achieved is as follows:

3.19.3 (10 mg/kg 2×wk)=74% inhibition; (p<0.001)

Gemcitabine (50 mg/kg q3dx2)=8% inhibition; (p<0.2)

Combination 3.19.3+Gemcitabine=88% inhibition (p<0.0003)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 8 b). These results demonstrate that combination treatment withanti-Ang2 antibody 3.19.3 and Gemcitabine leads to improvements inefficacy without additive toxicity in a pre-clinical model of coloncancer, and providing the basis for further clinical investigation ofthis combination.

Study 7.4. Determination of the Therapeutic efficacy of MonoclonalAntibody 3.19.3 in Combination with Docetaxel in Calu6 Xenograft Tumors

The anti-tumor activity of 3.19.3 was evaluated in combination withDocetaxel in the Calu6 xenograft model of lung cancer. Calu6 cells werecultured in flasks as routine until the cells reached sub-confluence.Cell suspensions containing approximately 3×10E6 cells were injectedsubcutaneously into the flank of female Swiss nude mice. When the tumorvolume reached 200 mm³, the mice were randomized in treatment groups of8-10 mice and the treatments were initiated. 3.19.3 (10 mg/kg) in salinewas injected intraperitoneally, twice per week for 2 weeks. Docetaxel(15 mg/kg) was administered by intravenous administration following aweekly schedule. The dimensions of each tumor and body weights weremeasured at least twice per week. The volume of the tumor was calculatedas: Volume=Length×(Width)×0.5 (cm³). As illustrated in FIG. 9 a, 3.19.3and Docetaxel delayed the growth of the Calu6 tumors with the singleagents. However the combination of 3.19.3 and Docetaxel had a greatereffect than the single agents alone as illustrated in FIG. 9 a. The %tumor growth inhibition achieved is as follows:

3.19.3 (10 mg/kg 2×wk)=20% inhibition; (p<0.12)

Docetaxel (15 mg/kg/week)=43% inhibition; (p<0.0007)

Combination 3.19.3+Docetaxel=71% inhibition (p<0.0001)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 9 b). These results demonstrate that combination treatment withanti-Ang2 antibody 3.19.3 and Docetaxel leads to improvements inefficacy without additive toxicity in a pre-clinical model of lungcancer, and providing the basis for further clinical investigation ofthis combination.

Study 7.5. Determination of the Therapeutic Efficacy of MonoclonalAntibody 3.19.3 in Combination with Oxaliplatin in H460 Xenograft Tumors

The anti-tumor activity of 3.19.3 was evaluated in combination withOxaliplatin in the H460 xenograft model of lung cancer. H460 cells werecultured in flasks as routine until the cells reached sub-confluence.Cell suspensions containing approximately 3×10E6 cells were injectedsubcutaneously into the flank of female Swiss nude mice. When the tumorvolume reached 200 mm³, the mice were randomized in treatment groups of8-10 mice and the treatments were initiated. 3.19.3 (10 mg/kg) in salinewas injected intraperitoneally, twice per week for 2 weeks. Oxaliplatin(5 mg/kg) was administered by intraperitoneal administration following aweekly schedule. The dimensions of each tumor and body weights weremeasured at least twice per week. The volume of the tumor was calculatedas: Volume=Length×(Width)×0.5 (cm³). As illustrated in FIG. 10 a, 3.19.3and Oxaliplatin delayed the growth of the H460 tumors with the singleagents. However the combination of 3.19.3 and Oxaliplatin had a greatereffect than the single agents alone as illustrated in FIG. 10 a. The %tumor growth inhibition achieved is as follows:

3.19.3 (10 mg/kg 2×wk)=67% inhibition; (p<0.001)

Oxaliplatin (5 mg/kg/week)=35% inhibition; (p<0.01)

Combination 3.19.3+Oxaliplatin=75% inhibition (p<0.0001)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 10 b). These results demonstrate that combination treatment withanti-Ang2 antibody 3.19.3 and Oxaliplatin leads to improvements inefficacy without additive toxicity in a pre-clinical model of lungcancer, and providing the basis for further clinical investigation ofthis combination.

Study 7.6. Determination of the Therapeutic Efficacy of MonoclonalAntibody 3.19.3 in Combination with the Mitotic Eg5 Inhibitor AZD4877 inH460 Xenograft Tumors

The anti-tumor activity of 3.19.3 was evaluated in combination withAZD4877 in the

H460 xenograft model of lung cancer. H460 cells were cultured in flasksas routine until the cells reached sub-confluence. Cell suspensionscontaining approximately 3×10E6 cells were injected subcutaneously intothe right flank of male NCr nu/nu mice. When the tumor volume reached200 mm³, the mice were randomized in treatment groups of 8-10 mice andthe treatments were initiated. 3.19.3 (10 mg/kg) in saline was injectedintraperitoneally, twice per week for 2 weeks. AZD4877 (10 mg/kg) wasadministered by intraperitoneal administration following a q4d schedule.The dimensions of each tumor and body weights were measured at leasttwice per week. The volume of the tumor was calculated as:Volume=Length×(Width)×0.5 (cm³). As illustrated in FIG. 11 a, 3.19.3 andAZD4877 delayed the growth of the H460 tumors with the single agents.However the combination of 3.19.3 and AZD4877 had a greater effect thanthe single agents alone as illustrated in FIG. 11 a. The % tumor growthinhibition achieved is as follows:

3.19.3 (10 mg/kg 2×wk)=64% inhibition; (p<0.001)

AZD4877 (10 mg/kg q4dx2)=50% inhibition; (p<0.001)

Combination 3.19.3+AZD4877=78% inhibition (p<0.0001)

No additional toxicity was observed with the combinations as compared tosingle-agent treatment alone as determined by changes in body weights(FIG. 11 b). These results demonstrate that combination treatment withanti-Ang2 antibody 3.19.3 and AZD4877 leads to improvements in efficacywithout additive toxicity in a pre-clinical model of lung cancer, andproviding the basis for further clinical investigation of thiscombination.

The studies of the anti Ang-2 antibody, 3.19.3 in combination withchemotherapeutic agents included Docetaxel, 5-flurouracil, Irinotecan,Oxaliplatin, or Gemcitabine which demonstrated at least additiveactivity with no increases in toxicity with the combinations asindicated by body weights. These results demonstrate that combinationtreatment with monoclonal antibody 3.19.3 and chemotherapy leads toimprovements in efficacy without additive toxicity in pre-clinicalmodels of cancer.

The results of the monoclonal antibody 3.19.3 xenograft combinationstudies with VEGF inhibitors and chemotherapeutic agents are summarizedin below:

TABLE 6 Summary of 3.19.3 with VEGF inhibitors and chemotherapeuticagents: Xenograft Chemotherapeutic Agent % Inhibition Significance(T-test) Lovo 5-flurouracil (100 mg/kg) 62% p < 0.02 3.19.3 (10 mg/kg)59% p < 0.09 Combination 85% p < 0.007 HT-29 Irinotecan (35 mg/kg) 56% p< 0.006 3.19.3 (10 mg/kg) 44% p < 0.0005 Combination 71% p < 0.0001Colo205 Gemcitabine (50 mg/kg)  8% p < 0.2 3.19.3 (10 mg/kg) 74% p <0.001 Combination 88% p < 0.0003 Calu6 Docetaxel (15 mg/kg) 43% p <0.0007 3.19.3 (10 mg/kg) 20% p < 0.12 Combination 71% p < 0.0001 H460Oxaliplatin (5 mg/kg) 35% p < 0.01 3.19.3 (10 mg/kg) 67% p < 0.001Combination 75% p < 0.0001

Example 8 Effect of Therapeutic Administration of the Ang-2 Antibody,3.19.3 on Disease Progression in the DBA/1 Murine Model of CollagenInduced Arthritis in vivo

Preparation of rat collagen type II emulsion: Bovine Collagen Type II(MD Biosciences, Cat #IMBII; Lot 090205) was stored at 4° C. in the darkuntil use. Prior to immunisation of animals, bovine collagen wasdissolved in 0.01 M acetic acid at a stock solution of 2 mg/mL andstored overnight in the dark at 4° C. On the day of immunisation,collagen was emulsified with equal volume of Freund's Complete Adjuvant(FCA [Difco, Cat #231131; Lot 850262/R1]) to give a solution at 1 mg/mL.

Induction of arthritis: On day 0, male DBA/1 mice (6-8 weeks old, HarlanSprague Dawley, UK) were lightly anaesthetised with 3.5% isoflurane andimmunized intra-dermal just above the root of the tail with 100 μg ratcollagen type II emulsified in FCA (1 mg/mL; 0.1 mL/mouse).

Staphylococcal Enterotoxin B (SEB) booster: On day 21, all mice wereanaesthetised as before and given a booster injection of 30 μg SEB (600μg/ml in water for injections [Toxin Technology, Cat #BT202; Lot 70903]emulsified in an equal volume of Freund's Incomplete Adjuvant [Sigma,Cat #F5506; Lot 112K8930] to give a final concentration of 300 μg/ml).50 μl×2 (equivalent to 30 μg SEB) is injected intra-dermal adjacent tothe immunisation site.

Assessment of arthritis: Clinical observations regarding welfare of theanimals were carried out daily at time of dosing. Observations forclinical signs of disease were carried out daily from day 20 postimmunisation, whereby the animals were removed from theirmicro-environment, and scored using the scoring system outlined below.

TABLE 7 Scoring system for hind and forepaws Clinical score Description0 Normal 1 Erythema and slight swelling of one of more toes. 2 Erythemaand obvious swelling of two or more toes, or mild swelling of the ankleor wrist without toe involvement. 3 Erythema and obvious swelling tosome toes and ankle or fore paw and wrist. 4 Erythema and severeswelling of ankle and digits.

Dosing: Animals were randomly assigned to treatment groups, as outlinedbelow.

TABLE 8 Treatment groups Group Treatment Number of animals 1 PBS Vehiclei.p 15   3.19.3 10 mg/kg i.p 15 3 Human IgG control antibody i.p 15 4Prednisolone 3 mg/kg p.o 10

Animals in treatment group 2 were therapeutically dosed intraperitonealy(i.p.) with 3.19.3 10 mL/kg every 3 days for 14 days from disease onset(defined as a clinical score of 2 in one or more paw). Purified humanIgG (hIgG) was used as a negative isotype control. Animals in treatmentgroup 4 were therapeutically dosed per oral (p.o.) with Prednisolone 3mg/kg daily for 14 days from disease onset (defined as a clinical scoreof 2 in one or more paw).

Termination: Animals were terminated 14 days post disease onset byexposure to rising concentration of carbon dioxide. Mouse paws wereexcised post mortem, fixed in 10% buffered formalin and decalcified.Decalcified paws were routinely processed and then embedded in paraffinblocks. Serial sections (10 μm) were cut and stained with hematoxylinand eosin for histologic analysis.

Data analysis: Area under the curve (AUC) for clinical diseaseprogression was calculated for each animal from disease onset. Unlessotherwise stated, statistical analysis was by one-way ANOVA withDunnett's post-hoc comparison to vehicle controls. P<0.05 was consideredto be statistically significant throughout the study.

Results: Significant reductions in both clinical signs of diseaseprogression (arthritic score) and histological assessment of synovitisand joint destruction were observed with 3.19.3 at a dose of 10 mg/kg(One-Way ANOVA with Dunnett's post-hoc comparison to vehicle control).

TABLE 9 Therapeutic effects of 3.19.3 on CIA disease progressionTreatment group Area Under Curve (AUC) % Inhibition PBS Vehicle i.p100.9 ± 10.9 — 3.19.3 10 mg/kg i.p 36.0 ± 8.7 64.3% ± 8.6% hIgG isotypecontrol i.p  92.4 ± 11.2  8.4% ± 11.1% Prednisolone 3 mg/kg p.o 24.5 ±9.6 76.1% ± 9.6%Table 9: Effect of therapeutic administration of 3.19.3 (10 mg/kg i.pevery 3 days) on clinical disease progression throughout the time courseof collagen-induced arthritis as measured by Area Under Curve [AUC](values represent means±standard error of the mean, n=15 for 3.19.3, PBSvehicle and hIgG treatment groups, n=10 for Prednisolone control group)

Histolopathological evaluation of CIA model treatment groups: Resultsshowed unequivocal evidence of an anti-arthritic effect followingadministration of 3.19.3—which morphologically was most evident uponsynovial hyperplasia and fibrosis. There were no atypical cell forms inthis study and no atypical presentation of bone. There is goodcorrelation between clinical score and histological measurements in thisstudy.

PBS Vehicle treatment group: Marked arthropathy was noted in themajority of animals treated with PBS vehicle. The pathology presentationwas a widespread—essentially mononuclear cell—synovitis invading thetibio-talus space and spreading towards the superficial facet of thecalcaneum. There was extensive lytic destruction of bone—notably thetalus and navicular bones—from both contact synovitis and from pannusexpansion into the bone marrow cavities and stromal cavities. Fibrosisof the synovium was common, with occasional fibrinoid deposits, withvariable degrees of synovial hyperplasia.

3.19.3 10 mg/kg treatment group: The 3.19.3 treatment group showed ahistologically significant reduction in the incidence and severity ofall arthropathy lesions, although it was most marked by reduction ofsynovial hyperplasia and fibrosis; together with a reduction in severityof both articular space and bone marrow localized pannus.

Human IgG isotype control treatment group: Histologically, there were nosignificant differences between this group and the PBS vehicle group.

Prednisolone 3 mg/kg treatment group: This group showed a markedreduction in the incidence and severity of all arthropathy lesions.

Summary: This study demonstrates that neutralization of angiopoietin-2has been shown to be efficacious in ameliorating collagen-inducedarthritis in male dba/1 mice (FIG. 12 a) with no significant change inmean body weight observed between each treatment group throughout theperiod from disease onset, suggesting that 3.19.3 therapy was welltolerated (FIG. 12 b). This study assessed efficacy of 3.19.3 at a doseof 10 mg/kg, and demonstrated reductions in both clinical signs ofdisease progression (arthritic score) and histological assessment ofsynovitis and joint destruction.

Example 9 Anti-Ang-2 Antibodies Inhibit Retinal Vascularization

The effect of anti-Ang-2 antibodies on retinal vascularization MEDI1/5antibodies was studied by comparing retinal samples from treated miceversus control treated mice.

Methods: CD1 pups are either left untreated or dosed intraperitoneallywith MEDI1/5 (1 mg/kg or 10 mg/kg) at p1, p3 and p5 (p1 being day ofbirth). At p10, pups were anesthetized with isoflurane and then perfusedwith 12.5 mg/ml FITC-dextran (Vector Labs). A small slit was made in thecornea and the entire eye was removed from the optic cup before placinginto 10% neutral buffered formalin. After 1 hour fixation in formalin,eyes were briefly rinsed in PBS and then placed in a dish of PBS fordissection. Retinas were carefully dissected and cut into a clover leafformation prior to mounting onto a glass slide with Vectashield (VectorLabs). Images of flatmounts were examined and acquired usingfluorescence microscopy (Nikon) with attached digital camera system.

Results: Retina vasculature in untreated pups (FIG. 13 a) and thosetreated with 0.3 mg/kg of MEDI1/5 (FIG. 13 b) extend to the outer edgeof retina, indicated with white arrow. Upon treatment with increasingdose of MEDI1/5, we see a dose response in the level of inhibition ofadvancing retina vessels (FIGS. 13 c and 13 d). The outer rim of retinais demarcated with dotted line and at both doses the hyloid vessels(white arrow) reach the outer rim of the retina but the retina vessels(white arrowheads) in 1 mg/kg MEDI1/5 (FIG. 13 c) approach closer to theperiphery of the retina in comparison to the 10 mg/kg MEDI1/5 (FIG. 13d) treated group. These results demonstrate a dose-dependent inhibitionof retinal angiogenesis by the treatment of MEDI1/5 anti-Ang-2antibodies.

Example 10 Anti-Ang-2 Antibodies Inhibit FGF-Mediated Angiogenesis

The anti-Ang-2 antibody, MEDI1/5 was evaluated for anti-angiogeniceffects in a FGF2 (basic FGF)-induced Matrigel™ plug assay. Recombinantmurine FGF2 (rmFGF basic; R&D Systems) was pre-mixed with Matrigel™(reduced growth factor, phenol red-free; Trevigen) at 1 μg/ml. Each 5-6week old female athymic mouse was subcutaneously implanted with 500 glof FGF2/Matrigel™ mixture. Antibody MEDI1/5 was administered 10 minutesprior to FGF2/Matrigel™ implantation and continued every three daysgiven intraperitoneal at 1, 10, and 20 mg/kg for a total of 3 doses. Theextent of angiogenesis was assessed after 10 days by measuring dextranfunctioning vessels. Mice were intervenously injected with 100 glFITC-Dextran (250,000MW; Sigma) at 25 mg/ml in saline. Twenty minutespost FITC-Dextran injection, mice are humanely euthanized and plugsdissected out. Plugs are then placed in lysing matrix tubes A (MPBiomedicals) containing 1 ml of PBS and homogenized on FastPrep machine(MP Biomedicals) for 60 seconds at 6.0 M/S. Samples were thencentrifuged at 10,000 rpm for 5 minutes and supernatant collected. 200μl of each sample (in duplicates) was then placed into a white, clearbottom 96 well plate and FITC output was read on EnVision instrument(Perkin Elmer).

For histology purposes, plugs were harvested and placed into 10%neutral-buffered formalin, processed and then paraffin embedded. Theparaffin embedded tissues were then sectioned and stained withhematoxylin and eosin.

Results: Approximately 0.78 μg of FITC-dextran could be detected inplugs induced with 1 μg/ml of FGF2 (FIG. 14 a). On the other hand, whenthese FGF2-treated plugs were exposed to 3 doses of MEDI1/5, rangingfrom 1, 10, and 20 mg/kg, the amount of FITC-dextran present in plugsharvested ranged from 0.05-3 μg. This significant reduction inFITC-dextran in MEDI1/5 treated animals suggests that MEDI1/5 inhibitsFGF2-induced angiogenesis in the Matrigel™ plugs. Plugs when stainedwith hematoxylin and eosin (FIG. 14 b) reveal that there are fewervessels in FGF2+MEDI1/5 plugs compared to FGF2 plugs alone, thus furtherproviding evidence that the anti Ang-2 antibody MEDI1/5 inhibitsFGF-mediated angiogenesis.

Example 11 Anti-Ang-2 Antibodies Inhibit Disease Progression in a MouseModel of Arthritis

In this Example, the Anti-Ang-2 antibody 3.19.3 was used in acollagen-induced arthritis (CIA) mouse model in an attempt todemonstrate therapeutic efficacy.

Materials and Methods: To study the effect of treatment on clinicaldisease progression in the collagen-induced arthritis (CIA) diseasemodel collagen-induced arthritis was induced in male DBA/1 mice andanimals dosed therapeutically with test treatments.

Preparation of rat collagen type II emulsion: Bovine Collagen Type II(MD Biosciences) was stored at 4° C. in the dark until use. Prior toimmunization of animals, bovine collagen was dissolved in 0.01 M aceticacid at a stock solution of 2 mg/mL and stored overnight in the dark at4° C. On the day of immunization, collagen was emulsified with equalvolume of Freund's Complete Adjuvant (FCA (Difco)) to give a solution at1 mg/mL.

Induction of arthritis: On day 0, male DBA/1 mice (6-8 weeks old, HarlanSprague Dawley, UK) were lightly anaesthetized with 3.5% isoflurane andimmunized intra-dermal just above the root of the tail with 100 μg ratcollagen type II emulsified in FCA (1 mg/mL; 0.1 mL/mouse).

Staphylococcal Enterotoxin B (SEB) booster: On day 21, all mice wereanaesthetised as before and given a booster injection of 30 μg SEB (600μg/ml in water for injections (Toxin Technology) emulsified in an equalvolume of Freund's Incomplete Adjuvant (Sigma) to give a finalconcentration of 300 μg/ml). 50 μl×2 (equivalent to 30 μg SEB) wasinject intradermal adjacent to the immunization site.

Assessment of arthritis: Clinical observations regarding welfare of theanimals were carried out daily at time of dosing. Observations forclinical signs of disease were carried out daily from day 20 postimmunization, whereby the animals were removed from theirmicro-environment, and scored using the scoring system outlined below.

TABLE 10 Scoring system for hind and forepaws Score Description 0 Normal1 Erythema and slight swelling of one of more toes 2 Erythema andobvious swelling of two or more toes, or mild swelling of the ankle orwrist without toe involvement 3 Erythema and obvious swelling to sometoes and ankle or fore paw and wrist 4 Erythema and severe swelling ofankle and digits

Dosing:

Animals were randomly assigned to treatment groups, as outlined in Table11 below.

TABLE 11 Treatment groups Group Treatment No. of animals 1 PBS Vehiclei.p. 20 2 0.1 mg/kg Antibody 3.19.3 20 3 1 mg/kg 3.19.3 i.p. 20 4 10mg/kg 3.19.3 i.p. 20 5 Human IgG control i.p. 20 6 Prednisolone 3 mg/kgp.o. 10

Animals in treatment groups 1-5 were therapeutically dosedintraperitoneally (i.p.) with 10 mL/kg every 3 days for 14 days fromdisease onset (defined as a clinical score of 2 in one or more paws).Purified human IgG (hIgG) was used as a negative isotype control.Animals in treatment group 6 were therapeutically dosed orally (p.o.)with Prednisolone 3 mg/kg daily for 14 days from disease onset (definedas a clinical score of 2 in one or more paws).

Termination: Animals were terminated 14 days post disease onset byexposure to rising concentration of carbon dioxide. Mouse paws wereexcised post mortem, fixed in 10% buffered formalin and decalcified.Decalcified paws were routinely processed and then embedded in paraffinblocks. Serial sections were cut and stained with hematoxylin and eosinfor histologic analysis, as well as CD31 staining for quantitation ofmicrovessel density in the synovium.

Results: Clinical Signs of Disease (Arthritis Score)

FIG. 15A shows the arthritic score mean (+/−standard error of the mean)against days from disease onset (i.e. days of treatment) (opensquares=PBS, open triangles=isotype control, closed squares=0.1 mg/kg3.19.3, closed triangles=1 mg/kg 3.19.3, closed circles=10 mg/kg 3.19.3and open circles=prednisolone). Dose-dependent reductions in clinicalsigns of disease progression (arthritic score) and were observed. Therewas a significant reduction at doses of 1 and 10 mg/kg. Area under thecurve (AUC) for clinical disease progression was calculated for eachanimal from disease onset (FIG. 15B), Table 12. Unless otherwise stated,statistical analysis was by one-way ANOVA with Dunnett's post-hoccomparison to vehicle controls. P<0.05 was considered to bestatistically significant throughout the study.

TABLE 12 AUC of Clinical Score of CIA mice treated with 3.19.3 TreatmentAUC % inhibition PBS Vehicle ip 118 +/− 10 Human IgG 10 mg/kg ip 102 +/−13 12% +/− 11% 3.19.3 0.1 mg/kg ip  99 +/− 15 16% +/− 13% 3.19.3 1 mg/kgip 46 +/− 9 53% +/− 7%* 3.19.3 10 mg/kg ip  67 +/− 11 44% +/− 9%*Prednisolone 3 mg/kg po  41 +/− 08 78% +/− 7%* *= P < 0.05 ANOVA - oneway, post hoc Dunnets v. vehicle

Histolopathological evaluation of CIA model treatment groups: Resultsshowed evidence of a dose-dependent anti-arthritic effect followingadministration of 3.19.3 on all parameters evaluated including synovialhyperplasmia (FIG. 15C), synovitis (FIG. 15D), pannus (FIG. 15E),synovial fibrosis (FIG. 15F), and periostitis (FIG. 15G).Histologically, there were no significant differences between theisotype control-treated group and the PBS vehicle group (FIGS. 15C-G).

Immunohistological evaluation of CD31+ (microvessel density) insynovium:Results showed significant reduction in microvessel density inthe synovium at doses of 1 and 10 mg/kg as well as with prednisolone.There was no effect with 0.1 mg/kg 3.19.3 treatment (FIG. 15H).

Overall Conclusions: This study demonstrates that neutralization ofangiopoietin-2 is efficacious in ameliorating collagen-induced arthritisin male DBA/1 mice. This study assessed efficacy of 3.19.3 at a dose of0.1, 1 and 10 mg/kg, and demonstrated dose-dependent reductions in bothclinical signs of disease progression (arthritic score) and histologicalassessment of synovitis and joint destruction, as well as microvesseldensity in the synovium. The prednisolone-treated group showed a markedreduction in the incidence and severity of all measures.

Example 12 Anti-Ang-2 Antibodies+Anti-TNFα Agents in Combination areEffective in a Prophylactic Model of Arthritis

In the Example, the anti-Ang-2 antibody MEDI1/5 as a standalone agent orin combination with the anti-TNFα agent ENBREL® was studied in aprophylactic model of arthritis. More specifically, the effect theMEDI1/5 antibody+/−ENBREL® on the clinical disease progression in theglucose 6 phosphate isomerase (G6PI) arthritis disease model.

Materials and Methods

Preparation of G6PI emulsion: On the day of immunization, FCA (Freund'scomplete adjuvant) was prepared by grinding M tuberculosis (Difco) intoIFA (Chondrex) to make a 1 mg/mL stock. Human G6PI was set to aconcentration of 3 mg/mL in phosphate buffered saline (Gibco). G6PI wasemulsified, by sonication, with equal volume of FCA to give an emulsionof 1.5 mg/mL.

Induction of arthritis: On day 0, male DBA/1J mice (9-10 week old,Jackson Laboratories) were administered 0.2 mL (300 μg G6PI) over twosites at the base of the tail via subcutaneous injections.

Assessment of arthritis: Observations for clinical signs of disease werecarried out daily from day 0 post immunization, whereby the animals wereremoved from their micro-environment, and scored using the scoringsystem outlined in Table 13 below.

TABLE 13 Scoring system for hind and forepaws Score Description 0 Normal0.5 Eruthema and slight swelling 1 Swelling of digits only 1.5 Localmild/moderate swelling 2 Severe local swelling of major joint 2.5 Severelocal swelling of major joint and other 3 Severe swelling of entire paw3.5 Severe swelling and partial or total ankylosis

Dosing: Animals were randomly assigned to treatment groups, as outlinedin Table 14 below.

TABLE 14 Treatment groups No. of Group Treatment animals 1 10 mg/kgHuman IgG1 control antibody i.p. 8 2 10 mg/kg MEDI1/5 i.p. 8 3 1 mg/kgENBREL ® i.p. 8 4 4 mg/kg ENBREL ® i.p. 8 5 10 mg/kg MEDI1/5 i.p + 1mg/kg ENBREL ® i.p. 8 6 10 mg/kg MEDI1/5 i.p + 1 mg/kg ENBREL ® i.p. 8

Animals in the enbrel treatment group were dosed daily with 10 mL/kgfrom days 0-9, then every 3 days until termination on day 16. All othertreatments were administered every 3 days.

Termination: Animals were terminated 16 days post immunization byexposure to rising concentration of carbon dioxide. Mouse paws wereexcised post mortem, fixed in 10% buffered formalin, assessed for bonemineral density and then decalcified. Decalcified paws were routinelyprocessed and then embedded in paraffin blocks. Serial sections were cutand stained with hematoxylin and eosin for histologic analysis. Thehistological analysis was scored by the following schedule:

-   -   Total joint scores        -   Both hind knee and ankle joints scored for:            -   Inflammation            -   Bone damage            -   Pannus formation            -   Cartilage damage        -   Score of 0-5            -   0=normal            -   1=minimal            -   2=mild            -   3=moderate            -   4=marked            -   5=severe

Bone Mineral Density

Bone mineral density was assessed in the stifle joint of the hind limbsusing DEXA imaging (GE Piximus). Following collection, hind limbs wereplaced in 10% neutral buffered saline for 4 days. Just prior to imaging,limbs were placed in 70% ethanol, then allowed to dry.

Results: Reductions in clinical signs of disease progression (arthriticscore) (FIGS. 16A (closed circles=isotype control, closed diamond=10mg/kg MEDI1/5,open diamond=1 mg/kg etanercept, grey diamond=combinationof 10 mg/kg MEDI1/5 with 1 mg/kg etanercept, open square=4 mg/kg enbrel,grey square=combination of 10 mg/kg MEDI1/5 with 4 mg/kg etanercept) and16B) were observed with either etanercept or MEDI1/5 treatment. Therewas a further reduction in clincal score when MEDI1/5 was administeredin combination with the lower dose of etanercept (One-Way ANOVA withDunnett's post-hoc comparison to isotype control). All joints in theisotype control antibody treated animals showed evidence of disease,whereas MEDI1/5 or 1 mg/kg etanercept treatments resulted in 31% or 22%of the joints showing no signs of disease, respectively. When the 10mg/kg MEDI1/5 and 1 mg/kg etanercept were administered in combination,50% of the joints of the animals in this group were diseasefree,comparable to the level of protection provided by the high dose (4mg/kg) of enbrel (53%) (Table 15). Histological assessment of synovitisand joint destruction (FIG. 16C) supported the clinical score results asdid the protection from loss of bone mineral density (FIG. 16D).

TABLE 15 Disease free joints observed in MEDI1/5 treated arthritic miceTreatment % Disease free joints 10 mg/ml Isotype control  0% 10 mg/kgMEDI1/5 31% 1 mg/kg etanercept 22% 10 mg/kg MEDI1/5 + 1 mg/kg etanercept50% 4 mg/kg etanercept 53% 10 mg/kg MEDI1/5 + 1 mg/kg etanercept 56%

Conclusions: This study demonstrates that neutralization ofangiopoietin-2 is efficacious in inhibiting the development ofG6PI-induced arthritis in male DBA/1J mice comparable to the anti-TNFcomparator, etanercept. This study also assessed efficacy of MEDI1/5combined with etanercept and demonstrated that combination treatmentwith the lower dose of enbrel provides further efficacy in both clinicalsigns of disease progression (arthritic score) and histologicalassessment of synovitis and joint destruction, as well as loss of bonemineral density, when administered prior to onset of disease.

Example 13 Anti-Ang-2 Antibodies+Anti-TNFα Agents in Combination areEffective in a Therapeutic Model of Arthritis

The effect of treatment with MEDI 1/5 with or without etanercept wasevaluated following onset of clinical disease in the glucose 6 phosphateisomerase (G6PI) arthritis disease model. G6PI-induced arthritis wasinduced in male DBA/1J mice and animals dosed therapeutically with testtreatments.

Materials and Methods: The mice in this study were prepared similarly aspresented in Example 12.

Dosing: This was a rolling admissions study: once an animal reached aclinical score of 3.5 to 5.0, it was randomly assigned to treatmentgroups (as outlined below) and dosing began. The day of initial dosingbecame study day 0 for that animal. Treatment groups were similar tothose presented in Example 12.

Termination: Study was terminated 12 days post treatment initiation. Nofurther endpoints were assessed on these animals.

Results: When administered in a therpeutic approach following the onsetof clinical disease, modest reductions in clinical signs of diseaseprogression (arthritic score) were observed with MEDI1/5 treatment,while both doses of etanercept tested had no effect on diseaseprogression. There was a more dramatic inhibition of progression ofdisease when MEDI1/5 (10 mg/kg) was administered in combination with thehigher dose of etanercept (4 mg/kg) (FIG. 17A (closed circles=isotypecontrol, closed diamond=MEDI1/5,open diamond=1 mg/kg etanercept, greydiamond=combination of MEDI1/5 with 1 mg/kg etanercept, open square=4mg/kg etanercept, grey square=combination of MEDI1/5 with 4 mg/kgetanercept).

Conclusions: This study demonstrates that neutralization ofangiopoietin-2 is efficacious in ameliorating G6PI-induced arthritis inmale DBA/1 mice comparable to the anti-TNF comparator, etanercept, whenadministered following the onset of clinical signs of disease. Thisstudy also assessed efficacy of MEDI1/5 combined with etanercept anddemonstrated that combination treatment provides enhanced efficacy overeither agent alone.

1-85. (canceled)
 86. An isolated antibody that binds to angiopoietin-2(Ang-2), wherein the antibody comprises a light chain variable regionof; a) SEQ ID No:3 (MEDI1); b) SEQ ID No:4 (MEDI2); c) SEQ ID No:5(MEDI3); d) SEQ ID No:6 (MEDI4); or e) SEQ ID No:8 (MEDI6).
 87. Theantibody according to claim 86, wherein the antibody comprises a lightchain variable region of SEQ ID No:3 (MEDI1).
 88. The antibody accordingto claim 86, wherein the antibody, when produced, exhibits a productionefficiency in a mammalian host cell equal to or greater than 2 times theproduction efficiency of an Ang-2 antibody comprising SEQ ID No: 1 andSEQ ID No: 2 (3.19.3).
 89. The antibody according to claim 86, whereinexhibits an aggregation rate that is at least 10% lower than theaggregation rate of an Ang-2 antibody comprising SEQ ID No: 1 and SEQ IDNo: 2 (3.19.3).
 90. The antibody according to claim 86, wherein exhibitsa melting temperature that is higher than the melting temperature of anAng-2 antibody comprising SEQ ID No: 1 and SEQ ID No: 2 (3.19.3). 91.The antibody according to claim 86, wherein the antibody furthercomprises a heavy chain variable region of SEQ ID No:7 (MEDI5).
 92. Theantibody according to claim 86, wherein the antibody comprises a lightchain variable region of SEQ ID No:3 (MEDI1) and a heavy chain variableregion of SEQ ID No:7 (MEDI5).
 93. An isolated nucleic acid encoding atleast one isolated antibody that binds to Ang-2 comprising a heavy andlight chain variable region of SEQ ID No:3 (MEDI1) and SEQ ID No:7(MEDI5).
 94. A pharmaceutical composition comprising: a) an isolatedantibody that binds to Ang-2, wherein the antibody comprises a lightchain variable region of SEQ ID No:3 (MEDI1) and a heavy chain variableregion of SEQ ID No:7 (MEDI5); and, b) an excipient.
 95. A method ofinhibiting angiogenesis of a cancer tumor in an animal in need thereof,wherein the method comprises administering to the animal a compositioncomprising; a) an isolated antibody that binds to Ang-2, wherein theantibody comprises a light chain variable region of SEQ ID No:3 (MEDI1)and a heavy chain variable region of SEQ ID No:7 (MEDI5); and, b) anexcipient.
 96. The method according to claim 95, wherein the compositionfurther comprises one or more other cancer therapeutic agents.
 97. Themethod according to claim 95, wherein the cancer is selected from thegroup consisting of melanoma, colon, colorectal, lung, small cell lungcarcinoma, non-small cell lung carcinoma, breast, rectum, stomach,glioma, prostate, ovary, testes, thyroid, kidney, liver, hepatocellularcarcinoma pancreas, brain, neck, glioblastoma, endometrial cancer, andcentral nervous system cancer.
 98. The method according to claim 95,wherein the composition is administered intravenously, subcutaneously,intratumorally, intramuscularly, parenterally, or orally.
 99. A methodof preventing, treating, or managing disease-related angiogenesis in ananimal in need thereof, wherein the method comprises administering acomposition comprising; a) an isolated antibody that binds to Ang-2;and, b) an excipient.
 100. The method according to claim 99, wherein thedisease-related angiogenesis is associated with seronegative arthritis,seropositive arthritis, arthritis related to other arthropathies,osteoarthritis or SLE.
 101. The method according to claim 100, whereinthe seropositive arthritis is rheumatoid arthritis.
 102. The methodaccording to claim 99, wherein the method further comprisesadministering one or more anti-inflammatory therapeutic agents.
 103. Themethod according to claim 102, wherein the anti-inflammatory therapeuticagent is a TNF-α antagonist.
 104. The method according to claim 99,wherein the antibody comprises a light chain variable region of SEQ IDNo:3 (MEDI1) and a heavy chain variable region of SEQ ID No:7 (MEDI5).